Anti-IL-20 antibodies and binding partners and methods of using in inflammation

ABSTRACT

The present invention relates to blocking the activity of IL-20 polypeptide molecules. IL-20 is a cytokine that is involved in inflammatory processes and human disease. IL-20RA/IL-20RB is a common receptor for IL-20. The present invention includes anti-IL-20 antibodies and binding partners, as well as methods for antagonizing IL-20 using such antibodies and binding partners.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/524,131, filed Nov. 21, 2003, and U.S. Provisional ApplicationSer. No. 60/555,857, filed Mar. 24, 2004, all of which are hereinincorporated by reference. Under 35 U.S.C. § 119(e)(1), this applicationclaims benefit of said Provisional Applications.

BACKGROUND OF THE INVENTION

Cytokines are soluble, small proteins that mediate a variety ofbiological effects, including the regulation of the growth anddifferentiation of many cell types (see, for example, Arai et al., Annu.Rev. Biochem. 59:783 (1990); Mosmann, Curr. Opin. Immunol. 3:311 (1991);Paul and Seder, Cell 76:241 (1994)). Proteins that constitute thecytokine group include interleukins, interferons, colony stimulatingfactors, tumor necrosis factors, and other regulatory molecules. Forexample, human interleukin-17 is a cytokine which stimulates theexpression of interleukin-6, intracellular adhesion molecule 1,interleukin-8, granulocyte macrophage colony-stimulating factor, andprostaglandin E2 expression, and plays a role in the preferentialmaturation of CD34+ hematopoietic precursors into neutrophils (Yao etal., J. Immunol. 155:5483 (1995); Fossiez et al., J. Exp. Med. 183:2593(1996)).

Receptors that bind cytokines are typically composed of one or moreintegral membrane proteins that bind the cytokine with high affinity andtransduce this binding event to the cell through the cytoplasmicportions of the certain receptor subunits. Cytokine receptors have beengrouped into several classes on the basis of similarities in theirextracellular ligand binding domains. For example, the receptor chainsresponsible for binding and/or transducing the effect of interferons aremembers of the class II cytokine receptor family, based upon acharacteristic 200 residue extracellular domain.

The demonstrated in vivo activities of cytokines and their receptorsillustrate the clinical potential of, and need for, other cytokines,cytokine receptors, cytokine agonists, and cytokine antagonists. Forexample, demonstrated in vivo activities of the pro-inflammatorycytokine family illustrates the enormous clinical potential of, and needfor antagonists of pro-inflammatory molecules.

The present invention addresses these needs by providing antibodies to apro-inflammatory cytokines, and particularly IL-20, including solubleIL-20RA, IL-20RB and IL-20RA/IL-20RB receptors and neutralizinganti-IL-20, IL-20RA and IL-20RB antibodies, as well as providing usestherefore in inflammatory disease, as well as related compositions andmethods.

DETAILED DESCRIPTION OF THE INVENTION

1. Overview

The present invention addresses these needs by providing antibodies topro-inflammatory cytokines and cytokine receptors. In particular, thepresent invention is directed to antibodies to IL-20 (referred tointerchangeably as Zcyto10) and one of its receptors, IL-20RA/IL-20RB(referred to interchangeably as ZcytoR7/pDIRS1), including neutralizinganti-IL-20 antibodies, anti-IL-20RA antibodies, anti-IL-20RB antibodies,and anti-IL-20RA/IL-20RB antibodies, as well as providing uses thereforein inflammatory disease, as well as related compositions and methods.

Amongst other inventions, the present invention provides novel uses forneutralizing antibodies to IL-20, and its receptor subunits IL-20RA andIL-20RB, as well as the receptor heterodimer, IL-20RA/IL-20RB.Specifically, these antibodies are useful in the treatment of humaninflammatory and autoimmune diseases. The present invention alsoprovides antibody fragments thereof, also for use in human inflammatoryand autoimmune diseases. The neutralizing anti-IL-20 antibodies,anti-IL-20RA antibodies, anti-IL-20RB antibodies, andanti-IL-20RA/IL-20RB heterodimer antibodies of the present invention,can be used to antagonize the activity of IL-20 in the treatment ofspecific human diseases such as psoriasis, psoriatic arthritis,arthritis, endotoxemia, inflammatory bowel disease (IBD), colitis, andother inflammatory conditions disclosed herein.

An illustrative nucleotide sequence that encodes human IL-20 is providedby SEQ ID NO:1. The corresponding encoded polypeptides are shown in SEQID NO:2. Analysis of a human cDNA clone encoding IL-20 (SEQ ID NO:1)revealed an open reading frame encoding 176 amino acids (SEQ ID NO:2),with the initial Met as shown in SEQ ID NO:1 and SEQ ID NO:2. It isbelieved that amino residues 1-24 are signal sequence, and the matureIL-20 polypeptide is represented by the amino acid sequence comprised ofresidues 25, a leucine through reside 176, a glutamic acid, also definedby SEQ ID NO:3. Another embodiment of the present invention is definedby the sequences of SEQ ID NO: 4 and SEQ ID NO: 5. The polypeptide ofSEQ ID NO: 5 is comprised of 151 amino acid residues wherein amino acids1-24 comprise a signal sequence and the mature sequence is comprised ofamino acid residues 25, a leucine, through amino acid 151 a glutamicacid, also defined by SEQ ID NO:6. Another active variant is comprisedof amino acid residues 33, a cysteine, through amino acid residue 176 ofSEQ ID NO:2. This variant is also defined by SEQ ID NO:7. Murine IL-20is encoded by SEQ ID NOs: 8-12. IL-20 is disclosed in commonly ownedU.S. Pat. No. 6,576,743, and commonly owned WIPO publication WO98/25228, both of which are incorporated herein by reference.

One receptor for IL-20 is comprised of two chains, an alpha chain and abeta chain. The alpha chain, hereinafter referred to as IL-20RA, wasformally called ZcytoR7. The beta chain, hereinafter referred to asIL-20RB, was formally called DIRS1. An illustrative nucleotide sequencefor IL-20RA is SEQ ID NO:13. The encoded polypeptide is shown in SEQ IDNO:14. Analysis of a human cDNA clone encoding IL-20RA (SEQ ID NO:13)revealed an open reading frame encoding 553 amino acids (SEQ ID NO:14)comprising an extracellular ligand-binding domain of approximately 221amino acid residues (residues 30-250 of SEQ ID NO:14 and SEQ ID NO:15),a transmembrane domain of approximately 24 amino acid residues (residues251-274 of SEQ ID NO:14), and an intracellular domain of approximately279 amino acid residues (residues 275-553 of SEQ ID NO:14). Thus, theextracellular domain of the human IL-20RA is comprised of a polypeptideselected from the group consisting of SEQ ID NOs: 16, 17, 18 and 19, thefull-length receptor subunit being comprised of SEQ ID NO:14. IL-20RA isdisclosed in commonly owned U.S. Pat. No. 5,945,511, and commonly ownedWIPO publication WO 98/03029, both of which are incorporated herein byreference.

An illustrative nucleotide sequence that encodes human IL-20RB (pDIRS1)is provided by SEQ ID NO:20. The encoded polypeptide is shown in SEQ IDNO:21. A variant IL-20RB is provided by SEQ ID NOs:22 and 23. Theextracellular domain of IL-20RB is comprised of a polypeptide selectedfrom the group consisting of SEQ ID NOs: 24, 25, 26, 27, 28, 29 and 30.IL-20RB is disclosed in U.S. Pat. No. 6,586,228, which is incorporatedherein by reference.

As described below, the present invention provides isolated polypeptidescomprising an amino acid sequence that is at least 70%, at least 80%, orat least 90%, or greater than 95%, such as 96%, 97%, 98%, or greaterthan 99% or more identical to SEQ ID NOS:2, 3, 14, 15, 21 or 23, whereinthe isolated polypeptide specifically binds with an antibody thatspecifically binds with a polypeptide consisting of the amino acidsequence of SEQ ID NOS:2, 3, 14, 15, 21 or 23. Moreover, the presentinvention also provides isolated polypeptides as disclosed above thatbind IL-20 (e.g., human IL-20 polypeptide sequence as shown in SEQ IDNO:2 or 3), IL-20RA (e.g., human IL-20RA polypeptide sequence as shownin SEQ ID NO:14 or 15), and IL-20RB (e.g., human IL-20RB polypeptidesequence as shown in SEQ ID NO:21 or 23). The human IL-20 polynucleotidesequence is shown in SEQ ID NO:1. The mouse IL-20 polynucleotidesequence is shown in SEQ ID NO: 10, and corresponding polypeptide isshown in SEQ ID NO:11.

The present invention also provides isolated polypeptides and epitopescomprising at least 15 contiguous amino acid residues of an amino acidsequence of SEQ ID NOS:2, 3, 14, 15, 21 or 23. Illustrative polypeptidesinclude polypeptides that either comprise, or consist of SEQ ID NOS:2,3, 14, 15, 21 or 23, an antigenic epitope thereof, or a functional IL-20binding fragment thereof. Moreover, the present invention also providesisolated polypeptides as disclosed above that bind, antagonize orneutralize the activity of IL-20.

The present invention also includes variant IL-20, IL-20RA and IL-20RBpolypeptides, wherein the amino acid sequence of the variant polypeptideshares an identity with the amino acid residues of SEQ ID NOS:2 or 3 forIL-20, SEQ ID NOS:14 or 15 for IL-20RA, or SEQ ID NOS:21 or 23 forIL-20RB, selected from the group consisting of at least 70% identity, atleast 80% identity, at least 90% identity, at least 95% identity, orgreater than 95% identity, such as 96%, 97%, 98%, or greater than 99% ormore identity, and wherein any difference between the amino acidsequence of the variant polypeptide and the corresponding amino acidsequence is due to one or more conservative amino acid substitutions.Such conservative amino acid substitutions are described herein.Moreover, the present invention also provides isolated polypeptides asdisclosed above that bind, antagonize or neutralize the activity ofIL-20.

The present invention further provides antibodies and antibody fragmentsthat specifically bind with such polypeptides. Exemplary antibodiesinclude neutralizing antibodies, polyclonal antibodies, murinemonoclonal antibodies, humanized antibodies derived from murinemonoclonal antibodies, and human monoclonal antibodies. Illustrativeantibody fragments include F(ab′)₂, F(ab)₂, Fab′, Fab, Fv, scFv, andminimal recognition units. Neutralizing antibodies preferably bindeither IL-20, IL-20RA or IL-20RB such that the interaction of IL-20 withits receptor is neutralized. The present invention further includescompositions comprising a carrier and a peptide, polypeptide, orantibody described herein.

In addition, the present invention provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and at least one ofsuch an expression vector or recombinant virus comprising suchexpression vectors. The present invention further includespharmaceutical compositions, comprising a pharmaceutically acceptablecarrier and a polypeptide or antibody described herein.

The present invention also contemplates anti-idiotype antibodies, oranti-idiotype antibody fragments, that specifically bind an antibody orantibody fragment that specifically binds a polypeptide comprising theamino acid sequence of SEQ ID NOs:2, 3, 5, 6, 7, 14-19, 21, 23-30 or afragment thereof. An exemplary anti-idiotype antibody binds with anantibody that specifically binds a polypeptide consisting of SEQ IDNO:3.

The present invention also provides fusion proteins, comprising eitheran IL-20, IL-20RA or IL-20RB polypeptide and an immunoglobulin moiety.In such fusion proteins, the immunoglobulin moiety may be animmunoglobulin heavy chain constant region, such as a human F_(c)fragment. The present invention further includes isolated nucleic acidmolecules that encode such fusion proteins.

The present invention also provides polyclonal and monoclonal antibodiescomprising either an IL-20 polypeptide fragment, or an IL-20RA orIL-20RB extracellular domain such as monomeric, homodimeric,heterodimeric and multimeric receptors, including soluble receptors.Moreover, such antibodies can be used antagonize the binding of IL-20 toits receptor.

Moreover, over expression of IL-20 was shown in human psoriatic lesions,suggesting that IL-20 is also involved in human psoriasis. Moreover, asdescribed herein, over expression of IL-20 in transgenic mice showedepidermal thickening and immune cell involvement indicative of apsoriatic phenotype. As such, antagonists to IL-20 activity, such asIL-20RA soluble receptors and antibodies thereto including eitheranti-human-IL-20, anti-human-IL-20RA, or anti-human-IL-20RB monoclonaland neutralizing antibodies of the present invention, are useful intherapeutic treatment of inflammatory diseases, particularly asantagonists to IL-20 in the treatment of psoriasis. Moreover,antagonists to IL-20 activity, such as IL-20RA soluble receptors andantibodies thereto including the anti-human-IL-20RA monoclonal andneutralizing antibodies of the present invention, are useful intherapeutic treatment of other inflammatory diseases for example asantagonists to IL-20 in the treatment of atopic dermatitis, IBD,colitis, Endotoxemia, arthritis, rheumatoid arthritis, and psoriaticarthritis adult respiratory disease (ARD), septic shock, multiple organfailure, inflammatory lung injury such as asthma or bronchitis,bacterial pneumonia, psoriasis, eczema, atopic and contact dermatitis,and inflammatory bowel disease such as ulcerative colitis and Crohn'sdisease.

These and other aspects of the invention will become evident uponreference to the following detailed description. In addition, variousreferences are identified below and are incorporated by reference intheir entirety.

2. Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (i.e., GAU and GAC triplets each encode Asp).

The term “structural gene” refers to a nucleic acid molecule that istranscribed into messenger RNA (mRNA), which is then translated into asequence of amino acids characteristic of a specific polypeptide.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

A “nucleic acid molecule construct” is a nucleic acid molecule, eithersingle- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

“Linear DNA” denotes non-circular DNA molecules having free 5′ and 3′ends. Linear DNA can be prepared from closed circular DNA molecules,such as plasmids, by enzymatic digestion or physical disruption.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclicAMP response elements (CREs), serum response elements (SREs; Treisman,Seminars in Cancer Biol. 1:47 (1990)), glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye etal., J. Biol. Chem. 269:25728 (1994)), SPI, cAMP response elementbinding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamerfactors (see, in general, Watson et al., eds., Molecular Biology of theGene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), andLemaigre and Rousseau, Biochem. J. 303:1 (1994)). If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter. Repressible promoters are also known.

A “core promoter” contains essential nucleotide sequences for promoterfunction, including the TATA box and start of transcription. By thisdefinition, a core promoter may or may not have detectable activity inthe absence of specific sequences that may enhance the activity orconfer tissue specific activity.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific,”“tissue-specific,” or “organelle-specific” manner.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage, that has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide tetracyclineresistance or ampicillin resistance.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector. In the presentcontext, an example of a recombinant host is a cell that produces IL-20,IL-20RA or IL-20RB from an expression vector. In contrast, IL-20,IL-20RA or IL-20RB can be produced by a cell that is a “natural source”of IL-20, IL-20RA or IL-20RB, and that lacks an expression vector.

“Integrative transformants” are recombinant host cells, in whichheterologous DNA has become integrated into the genomic DNA of thecells.

A “fusion protein” is a hybrid protein expressed by a nucleic acidmolecule comprising nucleotide sequences of at least two genes. Forexample, a fusion protein can comprise at least part of either an IL-20,IL-20RA or IL-20RB polypeptide fused with a polypeptide that binds anaffinity matrix. Such a fusion protein provides a means to isolate largequantities of these polypeptides using affinity chromatography.

As used herein, the term “antibody fusion protein” refers to arecombinant molecule that comprises an antibody component and atherapeutic agent. Examples of therapeutic agents suitable for suchfusion proteins include immunomodulators (“antibody-immunomodulatorfusion protein”) and toxins (“antibody-toxin fusion protein”).

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule termed a “ligand.” This interaction mediates theeffect of the ligand on the cell. Receptors can be membrane bound,cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormonereceptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor,erythropoietin receptor and IL-6 receptor). Membrane-bound receptors arecharacterized by a multi-domain structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. In certain membrane-boundreceptors, the extracellular ligand-binding domain and the intracellulareffector domain are located in separate polypeptides that comprise thecomplete functional receptor.

In general, the binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell, which in turnleads to an alteration in the metabolism of the cell. Metabolic eventsthat are often linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids.

A “soluble receptor” is a receptor polypeptide that is not bound to acell membrane. Soluble receptors are most commonly ligand-bindingreceptor polypeptides that lack transmembrane and cytoplasmic domains,and other linkage to the cell membrane such as via glycophosphoinositol(gpi). Soluble receptors can comprise additional amino acid residues,such as affinity tags that provide for purification of the polypeptideor provide sites for attachment of the polypeptide to a substrate, orimmunoglobulin constant region sequences. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis or translated from alternatively spliced mRNAs. Solublereceptors can be monomeric, homodimeric, heterodimeric, or multimeric,with multimeric receptors generally not comprising more than 9 subunits,preferably not comprising more than 6 subunits, and most preferably notcomprising more than 3 subunits. Receptor polypeptides are said to besubstantially free of transmembrane and intracellular polypeptidesegments when they lack sufficient portions of these segments to providemembrane anchoring or signal transduction, respectively. Solublereceptors of class I and class II cytokine receptors generally comprisethe extracellular cytokine binding domain free of a transmembrane domainand intracellular domain. It is well within the level of one of skill inthe art to delineate what sequences of a known class I or class IIcytokine sequence comprise the extracellular cytokine binding domainfree of a transmembrane domain and intracellular domain. Moreover, oneof skill in the art using the genetic code can readily determinepolynucleotides that encode such soluble receptor polypeptides.

The term “secretory signal sequence” denotes a DNA sequence that encodesa peptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure,such as 96%, 97%, or 98% or more pure, or greater than 99% pure. One wayto show that a particular protein preparation contains an isolatedpolypeptide is by the appearance of a single band following sodiumdodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the proteinpreparation and Coomassie Brilliant Blue staining of the gel. However,the term “isolated” does not exclude the presence of the samepolypeptide in alternative physical forms, such as dimers oralternatively glycosylated or derivatized forms.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a polypeptide encoded by asplice variant of an mRNA transcribed from a gene.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, co-stimulatory molecules, hematopoieticfactors, and the like, and synthetic analogs of these molecules.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity ofless than 10⁹ M⁻¹.

An “anti-idiotype antibody” is an antibody that binds with the variableregion domain of an immunoglobulin. In the present context, ananti-idiotype antibody binds with the variable region of an anti-IL-20antibody, and thus, an anti-idiotype antibody mimics an epitope ofIL-20. As a further example, an anti-idiotype antibody binds with thevariable region of an anti-IL-20RA antibody, and thus, an anti-idiotypeantibody mimics an epitope of IL-20RA. As yet another example, ananti-idiotype antibody binds with the variable region of an anti-IL-20RBantibody, and thus, an anti-idiotype antibody mimics an epitope ofIL-20RB.

An “antibody fragment” is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody. For example, an anti-IL-20 monoclonal antibody fragment bindswith an epitope of IL-20. As a further example, an anti-IL-20RAmonoclonal antibody fragment binds with an epitope of IL-20RA. As yetanother example, an anti-IL-20RB monoclonal antibody fragment binds withan epitope of IL-20RB.

The term “antibody fragment” also includes a synthetic or a geneticallyengineered polypeptide that binds to a specific antigen, such aspolypeptides consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

A “chimeric antibody” is a recombinant protein that contains thevariable domains and complementary determining regions derived from arodent antibody, while the remainder of the antibody molecule is derivedfrom a human antibody.

“Humanized antibodies” are recombinant proteins in which murinecomplementarity determining regions of a monoclonal antibody have beentransferred from heavy and light variable chains of the murineimmunoglobulin into a human variable domain. Construction of humanizedantibodies for therapeutic use in humans that are derived from murineantibodies, such as those that bind to or neutralize a human protein, iswithin the skill of one in the art.

As used herein, a “therapeutic agent” is a molecule or atom which isconjugated to an antibody moiety to produce a conjugate which is usefulfor therapy. Examples of therapeutic agents include drugs, toxins,immunomodulators, chelators, boron compounds, photoactive agents ordyes, and radioisotopes.

A “detectable label” is a molecule or atom which can be conjugated to anantibody moiety to produce a molecule useful for diagnosis. Examples ofdetectable labels include chelators, photoactive agents, radioisotopes,fluorescent agents, paramagnetic ions, or other marker moieties.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione Stransferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). DNA molecules encoding affinity tags areavailable from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

A “naked antibody” is an entire antibody, as opposed to an antibodyfragment, which is not conjugated with a therapeutic agent. Nakedantibodies include both polyclonal and monoclonal antibodies, as well ascertain recombinant antibodies, such as chimeric and humanizedantibodies.

As used herein, the term “antibody component” includes both an entireantibody and an antibody fragment.

An “immunoconjugate” is a conjugate of an antibody component with atherapeutic agent or a detectable label.

As used herein, the term “antibody fusion protein” refers to arecombinant molecule that comprises an antibody component and either anIL-20, IL-20RA or IL-20RB polypeptide component. Examples of an antibodyfusion protein include a protein that comprises a polypeptide domain ofIL-20 or an extracellular domain of either IL-20RA or IL-20RB, andeither an Fc domain or an antigen-binding region.

A “target polypeptide” or a “target peptide” is an amino acid sequencethat comprises at least one epitope, and that is expressed on a targetcell, such as a tumor cell, or a cell that carries an infectious agentantigen. T cells recognize peptide epitopes presented by a majorhistocompatibility complex molecule to a target polypeptide or targetpeptide and typically lyse the target cell or recruit other immune cellsto the site of the target cell, thereby killing the target cell.

An “antigenic peptide” is a peptide which will bind a majorhistocompatibility complex molecule to form an MHC-peptide complex whichis recognized by a T cell, thereby inducing a cytotoxic lymphocyteresponse upon presentation to the T cell. Thus, antigenic peptides arecapable of binding to an appropriate major histocompatibility complexmolecule and inducing a cytotoxic T cells response, such as cell lysisor specific cytokine release against the target cell which binds orexpresses the antigen. The antigenic peptide can be bound in the contextof a class I or class II major histocompatibility complex molecule, onan antigen presenting cell or on a target cell.

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A nucleic acid molecule can be designedto contain an RNA polymerase II template in which the RNA transcript hasa sequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an “anti-sense RNA” and a nucleic acid moleculethat encodes the anti-sense RNA is termed an “anti-sense gene.”Anti-sense RNA molecules are capable of binding to mRNA molecules,resulting in an inhibition of mRNA translation.

An “anti-sense oligonucleotide specific for IL-20” or a “IL-20anti-sense oligonucleotide” is an oligonucleotide having a sequence (a)capable of forming a stable triplex with a portion of the IL-20 gene, or(b) capable of forming a stable duplex with a portion of an mRNAtranscript of the IL-20 gene. An “anti-sense oligonucleotide specificfor IL-20RA” or a “IL-20RA anti-sense oligonucleotide” is anoligonucleotide having a sequence (a) capable of forming a stabletriplex with a portion of the IL-20RA gene, or (b) capable of forming astable duplex with a portion of an mRNA transcript of the IL-20RA gene.An “anti-sense oligonucleotide specific for IL-20RB” or a “IL-20RBanti-sense oligonucleotide” is an oligonucleotide having a sequence (a)capable of forming a stable triplex with a portion of the IL-20RB gene,or (b) capable of forming a stable duplex with a portion of an mRNAtranscript of the IL-20RB gene.

A “ribozyme” is a nucleic acid molecule that contains a catalyticcenter. The term includes RNA enzymes, self-splicing RNAs, self-cleavingRNAs, and nucleic acid molecules that perform these catalytic functions.A nucleic acid molecule that encodes a ribozyme is termed a “ribozymegene.”

An “external guide sequence” is a nucleic acid molecule that directs theendogenous ribozyme, RNase P, to a particular species of intracellularmRNA, resulting in the cleavage of the mRNA by RNase P. A nucleic acidmolecule that encodes an external guide sequence is termed an “externalguide sequence gene.”

The term “variant 1L-20 gene” refers to nucleic acid molecules thatencode a polypeptide having an amino acid sequence that is amodification of SEQ ID NO:2. Such variants include naturally-occurringpolymorphisms of IL-20 genes, as well as synthetic genes that containconservative amino acid substitutions of the amino acid sequence of SEQID NO:2. Additional variant forms of IL-20 genes are nucleic acidmolecules that contain insertions or deletions of the nucleotidesequences described herein. A variant IL-20 gene can be identified, forexample, by determining whether the gene hybridizes with a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, or itscomplement, under stringent conditions.

The term “variant J1L-20RA gene” refers to nucleic acid molecules thatencode a polypeptide having an amino acid sequence that is amodification of SEQ ID NO:11. Such variants include naturally-occurringpolymorphisms of IL-20RA genes, as well as synthetic genes that containconservative amino acid substitutions of the amino acid sequence of SEQID NO:11. Additional variant forms of IL-20RA genes are nucleic acidmolecules that contain insertions or deletions of the nucleotidesequences described herein. A variant IL-20RA gene can be identified,for example, by determining whether the gene hybridizes with a nucleicacid molecule having the nucleotide sequence of SEQ ID NO:10, or itscomplement, under stringent conditions.

The term “variant IL-20RB gene” refers to nucleic acid molecules thatencode a polypeptide having an amino acid sequence that is amodification of SEQ ID NO:13. Such variants include naturally-occurringpolymorphisms of IL-20RB genes, as well as synthetic genes that containconservative amino acid substitutions of the amino acid sequence of SEQID NO:13. Additional variant forms of IL-20RB genes are nucleic acidmolecules that contain insertions or deletions of the nucleotidesequences described herein. A variant IL-20RB gene can be identified,for example, by determining whether the gene hybridizes with a nucleicacid molecule having the nucleotide sequence of SEQ ID NO:12, or itscomplement, under stringent conditions.

Alternatively, variant 11-20, IL-20RA or IL-20RB genes can be identifiedby sequence comparison. Two amino acid sequences have “100% amino acidsequence identity” if the amino acid residues of the two amino acidsequences are the same when aligned for maximal correspondence.Similarly, two nucleotide sequences have “100% nucleotide sequenceidentity” if the nucleotide residues of the two nucleotide sequences arethe same when aligned for maximal correspondence. Sequence comparisonscan be performed using standard software programs such as those includedin the LASERGENE bioinformatics computing suite, which is produced byDNASTAR (Madison, Wis.). Other methods for comparing two nucleotide oramino acid sequences by determining optimal alignment are well-known tothose of skill in the art (see, for example, Peruski and Peruski, TheInternet and the New Biology: Tools for Genomic and Molecular Research(ASM Press, Inc. 1997), Wu et al. (eds.), “Information Superhighway andComputer Databases of Nucleic Acids and Proteins,” in Methods in GeneBiotechnology, pages 123-151 (CRC Press, Inc. 1997), and Bishop (ed.),Guide to Human Genome Computing, 2nd Edition (Academic Press, Inc.1998)). Particular methods for determining sequence identity aredescribed below.

Regardless of the particular method used to identify any of the variantgenes or variant polypeptides described above, a variant gene orpolypeptide encoded by a variant gene may be functionally characterizedby its ability to bind specifically to an anti-IL-20, anti-IL-20RA oranti-IL-20RB antibody. A variant IL-20RA or IL-20RB gene or variantpolypeptide may also be functionally characterized the ability to bindto its ligand, IL-20, using a biological or biochemical assay describedherein.

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

The present invention includes functional fragments of any of the IL-20,IL-20RA or IL-20RB genes. For example, within the context of thisinvention, a “functional fragment” of a IL-20RA gene refers to a nucleicacid molecule that encodes a portion of a IL-20RA polypeptide which is adomain described herein or at least specifically binds with ananti-IL-20RA antibody.

Due to the imprecision of standard analytical methods, molecular weightsand lengths of polymers are understood to be approximate values. Whensuch a value is expressed as “about” X or “approximately” X, the statedvalue of X will be understood to be accurate to ±10%.

3. Production of IL-20, IL-20RA and Il-20RB Polynucleotides or Genes

Nucleic acid molecules encoding a human IL-20, IL-20RA or IL-20RB genecan be obtained by screening a human cDNA or genornic library usingpolynucleotide probes based upon SEQ ID NO:1, SEQ ID NO:13 or SEQ IDNO:20, respectively. These techniques are standard and well-established,and may be accomplished using cloning kits available by commercialsuppliers. See, for example, Ausubel et al. (eds.), Short Protocols inMolecular Biology, 3^(rd) Edition, John Wiley & Sons 1995; Wu et al.,Methods in Gene Biotechnology, CRC Press, Inc. 1997; Aviv and Leder,Proc. Nat'l Acad. Sci. USA 69:1408 (1972); Huynh et al., “Constructingand Screening cDNA Libraries in λgt10 and λgt11,” in DNA Cloning: APractical Approach Vol. I, Glover (ed.), page 49 (IRL Press, 1985); Wu(1997) at pages 47-52.

Nucleic acid molecules that encode a human IL-20, IL-20RA or IL-20RBgene can also be obtained using the polymerase chain reaction (PCR) witholigonucleotide primers having nucleotide sequences that are based uponthe nucleotide sequences of any of the genes or cDNA. General methodsfor screening libraries with PCR are provided by, for example, Yu etal., “Use of the Polymerase Chain Reaction to Screen Phage Libraries,”in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methodsand Applications, White (ed.), Humana Press, Inc., 1993. Moreover,techniques for using PCR to isolate related genes are described by, forexample, Preston, “Use of Degenerate Oligonucleotide Primers and thePolymerase Chain Reaction to Clone Gene Family Members,” in Methods inMolecular Biology, Vol. 15: PCR Protocols: Current Methods andApplications, White (ed.), Humana Press, Inc. 1993. As an alternative, aIL-20RA gene can be obtained by synthesizing nucleic acid moleculesusing mutually priming long oligonucleotides and the nucleotidesequences described herein (see, for example, Ausubel (1995)).Established techniques using the polymerase chain reaction provide theability to synthesize DNA molecules at least two kilobases in length(Adang et al., Plant Molec. Biol. 21:1131 (1993), Bambot et al., PCRMethods and Applications 2:266 (1993), Dillon et al., “Use of thePolymerase Chain Reaction for the Rapid Construction of SyntheticGenes,” in Methods in Molecular Biology, Vol. 15: PCR Protocols: CurrentMethods and Applications, White (ed.), pages 263-268, (Humana Press,Inc. 1993), and Holowachuk et al., PCR Methods Appl. 4:299 (1995)). Forreviews on polynucleotide synthesis, see, for example, Glick andPasternak, Molecular Biotechnology, Principles and Applications ofRecombinant DNA (ASM Press 1994), Itakura et al., Annu. Rev. Biochem.53:323 (1984), and Climie et al., Proc. Nat'l Acad. Sci. USA 87:633(1990).

4. Production of IL-20, IL-20RA or IL-20RB Gene Variants

The present invention provides a variety of nucleic acid molecules,including DNA and RNA molecules, that encode the IL-20, IL-20RA orIL-20RB polypeptides disclosed herein. Those skilled in the art willreadily recognize that, in view of the degeneracy of the genetic code,considerable sequence variation is possible among these polynucleotidemolecules. Moreover, the present invention also provides isolatedsoluble monomeric, homodimeric, heterodimeric and multimeric receptorpolypeptides that comprise at least one IL-20RA or IL-20RB receptorsubunit that is substantially homologous to the receptor polypeptide ofSEQ ID NO:11 or SEQ ID NO:13, respectively.

Table 1 sets forth the one-letter codes to denote degenerate nucleotidepositions. “Resolutions” are the nucleotides denoted by a code letter.“Complement” indicates the code for the complementary nucleotide(s). Forexample, the code Y denotes either C or T, and its complement R denotesA or G, A being complementary to T, and G being complementary to C.TABLE 1 Nucleotide Resolution Complement Resolution A A T T C C G G G GC C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|GW A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T HA|C|T N A|C|G|T N A|C|G|T

The degenerate codons, encompassing all possible codons for a givenamino acid, are set forth in Table 2. TABLE 2 One Amino LetterDegenerate Acid Code Codons Codon Cys C TGC TGT TGY Ser S AGC AGT TCATCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro P CCA CCC CCG CCT CCN AlaA GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp DGAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg RAGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met M ATG ATG Ile I ATAATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTNPhe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGG TGG Ter · TAA TAG TGA TRRAsn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding an amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequences of IL-20 (SEQ ID NO:2), IL-20RA (SEQ ID NO:11) and IL-20RB(SEQ ID NO:13). Variant sequences can be readily tested forfunctionality as described herein.

Different species can exhibit “preferential codon usage.” In general,see, Grantham et al., Nucl. Acids Res. 8:1893 (1980), Haas et al. Curr.Biol. 6:315 (1996), Wain-Hobson et al., Gene 13:355 (1981), Grosjean andFiers, Gene 18:199 (1982), Holm, Nuc. Acids Res. 14:3075 (1986),Ikemura, J. Mol. Biol. 158:573 (1982), Sharp and Matassi, Curr. Opin.Genet. Dev. 4:851 (1994), Kane, Curr. Opin. Biotechnol. 6:494 (1995),and Makrides, Microbiol. Rev. 60:512 (1996). As used herein, the term“preferential codon usage” or “preferential codons” is a term of artreferring to protein translation codons that are most frequently used incells of a certain species, thus favoring one or a few representativesof the possible codons encoding each amino acid (See Table 2). Forexample, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG,or ACT, but in mammalian cells ACC is the most commonly used codon; inother species, for example, insect cells, yeast, viruses or bacteria,different Thr codons may be preferential. Preferential codons for aparticular species can be introduced into the polynucleotides of thepresent invention by a variety of methods known in the art. Introductionof preferential codon sequences into recombinant DNA can, for example,enhance production of the protein by making protein translation moreefficient within a particular cell type or species. Therefore, thedegenerate codon sequences disclosed herein serve as a template foroptimizing expression of polynucleotides in various cell types andspecies commonly used in the art and disclosed herein. Sequencescontaining preferential codons can be tested and optimized forexpression in various species, and tested for functionality as disclosedherein.

Any of the IL-20, IL-20RA or IL-20RB-encoding cDNA can be isolated by avariety of methods, such as by probing with a complete or partial humancDNA or with one or more sets of degenerate probes based on thedisclosed sequences. A cDNA can also be cloned using the polymerasechain reaction with primers designed from the representative humansequences disclosed herein. In addition, a cDNA library can be used totransform or transfect host cells, and expression of the cDNA ofinterest can be detected with an antibody to any of the IL-20, IL-20RAor IL-20RB polypeptides.

Those skilled in the art will recognize that the sequences disclosed inSEQ ID NOS:1, 10 and 12 represent a single allele of human IL-20,IL-20RA or L-20RB, and that allelic variation and alternative splicingare expected to occur. Allelic variants of any of these sequences can becloned by probing cDNA or genomic libraries from different individualsaccording to standard procedures. Allelic variants of the nucleotidesequences disclosed herein, including those containing silent mutationsand those in which mutations result in amino acid sequence changes, arewithin the scope of the present invention, as are proteins which areallelic variants of the amino acid sequences disclosed herein. cDNAmolecules generated from alternatively spliced mRNAs, which retain theproperties of any of the polypeptides referenced above are includedwithin the scope of the present invention, as are polypeptides encodedby such cDNAs and mRNAs. Allelic variants and splice variants of thesesequences can be cloned by probing cDNA or genomic libraries fromdifferent individuals or tissues according to standard procedures knownin the art.

Using the methods discussed above, one of ordinary skill in the art canprepare a variety of polypeptides that comprise IL-20 or a fragmentthereof that is substantially homologous to SEQ ID NO:1. One of ordinaryskill in the art could also prepare a variety of polypeptides thatcomprise either a soluble I1L-20RA receptor subunit that issubstantially homologous to SEQ ID NO:10, or that encodes amino acids ofSEQ ID NO:11, or a soluble IL-20RB receptor subunit that issubstantially homologous to SEQ ID NO:12, or that encodes amino acids ofSEQ ID NO:13, or allelic variants of either, and all of which retain theligand-binding properties of the wild-type IL-20RA or IL-20RB receptorsubunit. Such polypeptides may also include additional polypeptidesegments as generally disclosed herein.

Within certain embodiments of the invention, the isolated nucleic acidmolecules can hybridize under stringent conditions to nucleic acidmolecules comprising nucleotide sequences disclosed herein. For example,such nucleic acid molecules can hybridize under stringent conditions tonucleic acid molecules comprising the nucleotide sequence of any of SEQID NO:1, SEQ ID NO:13 or SEQ ID NO:20 or to nucleic acid moleculescomprising a nucleotide sequence complementary these sequences, orfragments thereof.

In general, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe. Following hybridization, thenucleic acid molecules can be washed to remove non-hybridized nucleicacid molecules under stringent conditions, or under highly stringentconditions. See, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition (Cold Spring Harbor Press 1989);Ausubel et al., (eds.), Current Protocols in Molecular Biology (JohnWiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to MolecularCloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev.Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis software such asOLIGO 6.0 (LSR; Long Lake, Minn.) and Primer Premier 4.0 (PremierBiosoft International; Palo Alto, Calif.), as well as sites on theInternet, are available tools for analyzing a given sequence andcalculating T_(m) based on user-defined criteria. It is well within theabilities of one skilled in the art to adapthybridization and washconditions for use with a particular polynucleotide hybrid.

The present invention also provides isolated IL-20, IL-20RA and IL-20RBpolypeptides that have a substantially similar sequence identity to thepolypeptides of SEQ ID NO:2, SEQ ID NO:11 and SEQ ID NO:13, or theirorthologs. The term “substantially similar sequence identity” is usedherein to denote polypeptides having at least 70%, at least 80%, atleast 90%, at least 95%, such as 96%, 97%, 98%, or greater than 95%sequence identity to the sequences shown in SEQ ID NO:2, SEQ ID NO:11and SEQ ID NO: 13, or their orthologs. For example, variant andorthologous IL-20RA receptors can be used to generate an immune responseand raise cross-reactive antibodies to human IL-20RA. Such antibodiescan be humanized, and modified as described herein, and usedtherapeutically to treat psoriasis, psoriatic arthritis, IBD, colitis,endotoxemia as well as in other therapeutic applications describedherein.

The present invention also contemplates IL-20, IL-20RA and IL-20RBvariant nucleic acid molecules that can be identified using twocriteria: a determination of the similarity between the encodedpolypeptide with the amino acid sequence of SEQ ID NO:2, SEQ ID NO:11and SEQ ID NO:13, and a hybridization assay. Such variants includenucleic acid molecules (1) that remain hybridized with a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:10 andSEQ ID NO:12 (or their complements) under stringent washing conditions,in which the wash stringency is equivalent to 0.5×-2×SSC with 0.1% SDSat 55-65° C., and (2) that encode a polypeptide having at least 70%, atleast 80%, at least 90%, at least 95%, or greater than 95% such as 96%,97%, 98%, or 99%, sequence identity to the amino acid sequence of SEQ IDNO:3. Alternatively, these variants can be characterized as nucleic acidmolecules (1) that remain hybridized with a nucleic acid molecule havingthe nucleotide sequence of SEQ ID NO:1, SEQ ID NO:10 and SEQ ID NO:12(or their complement) under highly stringent washing conditions, inwhich the wash stringency is equivalent to 0.1×-0.2×SSC with 0.1% SDS at50-65° C., and (2) that encode a polypeptide having at least 70%, atleast 80%, at least 90%, at least 95% or greater than 95%, such as 96%,97%, 98%, or 99% or greater, sequence identity to the amino acidsequence of any of SEQ ID NO:2, SEQ ID NO:11 and SEQ ID NO:13.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 3 (amino acids are indicated by the standard one-lettercodes). The percent identity is then calculated as: ([Total number ofidentical matches]/[length of the longer sequence plus the number ofgaps introduced into the longer sequence in order to align the twosequences])(100). TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R−1 5 N −2 0 6 D −2 −2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 25 G 0 −2 0 −1 −3 −2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3−4 −3 4 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −25 M −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0−3 0 6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0−1 −2 −2 0 −1 −2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W−3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1−2 −3 2 −1 −1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1−1 −2 −2 0 −3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativeIL-20, IL-20RA or IL-20RB variant. The FASTA algorithm is described byPearson and Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and byPearson, Meth. Enzymol. 183:63 (1990). Briefly, FASTA firstcharacterizes sequence similarity by identifying regions shared by thequery sequence and a test sequence that have either the highest densityof identities (if the ktup variable is 1) or pairs of identities (ifktup=2), without considering conservative amino acid substitutions,insertions, or deletions. The ten regions with the highest density ofidentities are then rescored by comparing the similarity of all pairedamino acids using an amino acid substitution matrix, and the ends of theregions are “trimmed” to include only those residues that contribute tothe highest score. If there are several regions with scores greater thanthe “cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Illustrative parametersfor FASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdescribed above.

The present invention includes nucleic acid molecules that encode apolypeptide having a conservative amino acid change, compared with anamino acid sequence disclosed herein. For example, variants can beobtained that contain one or more amino acid substitutions of any of thesequences disclosed herein, in which an alkyl amino acid is substitutedfor an alkyl amino acid, an aromatic amino acid is substituted for anaromatic amino acid, a sulfur-containing amino acid is substituted for asulfur-containing amino acid, a hydroxy-containing amino acid issubstituted for a hydroxy-containing amino acid, an acidic amino acid issubstituted for an acidic amino acid, a basic amino acid is substitutedfor a basic amino acid, or a dibasic monocarboxylic amino acid issubstituted for a dibasic monocarboxylic amino acid. Among the commonamino acids, for example, a “conservative amino acid substitution” isillustrated by a substitution among amino acids within each of thefollowing groups: (1) glycine, alanine, valine, leucine, and isoleucine,(2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)lysine, arginine and histidine. The BLOSUM62 table is an amino acidsubstitution matrix derived from about 2,000 local multiple alignmentsof protein sequence segments, representing highly conserved regions ofmore than 500 groups of related proteins (Henikoff and Henikoff, Proc.Nat'l Acad. Sci. USA 89:10915 (1992)). Accordingly, the BLOSUM62substitution frequencies can be used to define conservative amino acidsubstitutions that may be introduced into the amino acid sequences ofthe present invention. Although it is possible to design amino acidsubstitutions based solely upon chemical properties (as discussedabove), the language “conservative amino acid substitution” preferablyrefers to a substitution represented by a BLOSUM62 value of greater than−1. For example, an amino acid substitution is conservative if thesubstitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.According to this system, preferred conservative amino acidsubstitutions are characterized by a BLOSUM62 value of at least 1 (e.g.,1, 2 or 3), while more preferred conservative amino acid substitutionsare characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).

Particular variants of any of IL-20, IL-20RA or IL-20RB arecharacterized by having at least 70%, at least 80%, at least 90%, atleast 95% or greater than 95% such as 96%, 97%, 98%, or 99% or greatersequence identity to the corresponding amino acid sequences (e.g., SEQID NO:2, SEQ ID NO:14 and SEQ IfD NO:21), wherein the variation in aminoacid sequence is due to one or more conservative amino acidsubstitutions.

Conservative amino acid changes in any of the IL-20, IL-20RA or L-20RBgenes can be introduced, for example, by substituting nucleotides forthe nucleotides recited in any of SEQ ID NO:1, SEQ ID NO:13 and SEQ IDNO:20. Such “conservative amino acid” variants can be obtained byoligonucleotide-directed mutagenesis, linker-scanning mutagenesis,mutagenesis using the polymerase chain reaction, and the like (seeAusubel (1995); and McPherson (ed.), Directed Mutagenesis: A PracticalApproach (IRL Press 1991)). A variant IL-20, IL-20RA or IL-20RBpolypeptide can be identified by the ability to specifically bind to itsrespective antibody.

The proteins of the present invention can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is typicallycarried out in a cell-free system comprising an E. coli S30 extract andcommercially available enzymes and other reagents. Proteins are purifiedby chromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722 (1991), Ellman et al., Methods Enzymol. 202:301 (1991), Chunget al., Science 259:806 (1993), and Chung et al., Proc. Nat'l Acad. Sci.USA 90:10145 (1993).

In a second method, translation is carried out in Xenopus oocytes bymicroinjection of mutated mRNA and chemically aminoacylated suppressortRNAs (Turcatti et al., J. Biol. Chem. 271:19991 (1996)). Within a thirdmethod, E. coli cells are cultured in the absence of a natural aminoacid that is to be replaced (e.g., phenylalanine) and in the presence ofthe desired non-naturally occurring amino acid(s) (e.g.,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or4-fluorophenylalanine). The non-naturally occurring amino acid isincorporated into the protein in place of its natural counterpart. See,Koide et al., Biochem. 33:7470 (1994). Naturally occurring amino acidresidues can be converted to non-naturally occurring species by in vitrochemical modification. Chemical modification can be combined withsite-directed mutagenesis to further expand the range of substitutions(Wynn and Richards, Protein Sci. 2:395 (1993)).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for any of the IL-20,IL-20RA or IL-20RB amino acid residues.

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Nat'l Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity to identify amino acid residues that are critical to theactivity of the molecule. See also, Hilton et al., J. Biol. Chem.271:4699 (1996).

Although sequence analysis can be used to further define the IL-20binding domain or the IL-20RA or IL-20RB ligand binding region, aminoacids that play a role in IL-20, IL-20RA and IL-20RB binding activity(such as binding to ligand IL-20, or to an anti-IL-20RA or IL-20RBantibody) can also be determined by physical analysis of structure, asdetermined by such techniques as nuclear magnetic resonance,crystallography, electron diffraction or photoaffinity labeling, inconjunction with mutation of putative contact site amino acids. See, forexample, de Vos et al., Science 255:306 (1992), Smith et al., J. Mol.Biol. 224:899 (1992), and Wlodaver et al., FEBS Lett. 309:59 (1992).

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53 (1988)) or Bowie and Sauer(Proc. Nat'l Acad. Sci. USA 86:2152 (1989)). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner etal., U.S. Pat. No. 5,223,409, Huse, international publication No. WO92/06204, and region-directed mutagenesis (Derbyshire et al., Gene46:145 (1986), and Ner et al., DNA 7.127, (1988)). Moreover, IL-20RAlabeled with biotin or FITC can be used for expression cloning ofIL-20RA ligands.

Variants of the disclosed IL-20, IL-20RA and IL-20RB nucleotide andpolypeptide sequences can also be generated through DNA shuffling asdisclosed by Stemmer, Nature 370:389 (1994), Stemmer, Proc. Nat'l Acad.Sci. USA 91:10747 (1994), and international publication No. WO 97/20078.Briefly, variant DNA molecules are generated by in vitro homologousrecombination by random fragmentation of a parent DNA followed byreassembly using PCR, resulting in randomly introduced point mutations.This technique can be modified by using a family of parent DNAmolecules, such as allelic variants or DNA molecules from differentspecies, to introduce additional variability into the process. Selectionor screening for the desired activity, followed by additional iterationsof mutagenesis and assay provides for rapid “evolution” of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode biologically active polypeptides, or polypeptidesthat bind with anti-IL-20, anti-IL-20RA or anti-IL-20RB antibodies, canbe recovered from the host cells and rapidly sequenced using modernequipment. These methods allow the rapid determination of the importanceof individual amino acid residues in a polypeptide of interest, and canbe applied to polypeptides of unknown structure.

The present invention also includes “functional fragments” of IL-20,IL-20RA and IL-20RB polypeptides and nucleic acid molecules encodingsuch functional fragments. Routine deletion analyses of nucleic acidmolecules can be performed to obtain functional fragments of a nucleicacid molecule that encodes an IL-20, IL-20RA or IL-20RB polypeptide. Asan illustration, DNA molecules having the nucleotide sequence of SEQ IDNO:1 (IL-20) can be digested with Bal31 nuclease to obtain a series ofnested deletions. The fragments are then inserted into expressionvectors in proper reading frame, and the expressed polypeptides areisolated and tested for the ability to bind anti-IL-20 antibodies. Onealternative to exonuclease digestion is to use oligonucleotide-directedmutagenesis to introduce deletions or stop codons to specify productionof a desired fragment. Alternatively, particular fragments of an IL-20gene can be synthesized using the polymerase chain reaction.

This general approach is exemplified by studies on the truncation ateither or both termini of interferons have been summarized byHorisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993),Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987), Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation, Vol. I, Boynton etal., (eds.) pages 169-199 (Academic Press 1985), Coumailleau et al., J.Biol. Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291(1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995), and Meiselet al., Plant Molec. Biol. 30:1(1996).

The present invention also contemplates functional fragments of anIL-20, IL-20RA or IL-20RB gene that have amino acid changes, comparedwith an amino acid sequence disclosed herein. Such a variant gene can beidentified on the basis of structure by determining the level ofidentity with disclosed nucleotide and amino acid sequences, asdiscussed above. An alternative approach to identifying a variant geneon the basis of structure is to determine whether a nucleic acidmolecule encoding a potential variant IL-20, IL-20RA or IL-20RB gene canhybridize to a nucleic acid molecule comprising a nucleotide sequence,such as SEQ ID NO:1, SEQ ID NO:13 or SEQ ID NO:20, respectively.

The present invention also includes using functional fragments of IL-20,IL-20RA or IL-20RB polypeptides, antigenic epitopes, epitope-bearingportions of IL-20, IL-20RA or IL-20RB polypeptides, and nucleic acidmolecules that encode such functional fragments, antigenic epitopes, andepitope-bearing portions of IL-20, IL-20RA or IL-20RB polypeptides. Suchfragments are used to generate polypeptides for use in generatingantibodies and binding partners that bind, block, reduce, antagonize orneutralize activity of IL-20. A “functional” IL-20 polypeptide orfragment thereof as defined herein is characterized by its ability toantagonize IL-20 inflammatory, proliferative or differentiatingactivity, by its ability to induce or inhibit specialized cellfunctions, or by its ability to bind specifically to an anti-IL-20antibody, cell, or IL-20 receptor. A “functional” IL-20RA polypeptide orfragment thereof as defined herein is characterized by its ability toantagonize IL-20 inflammatory, proliferative or differentiatingactivity, by its ability to induce or inhibit specialized cellfunctions, or by its ability to bind specifically to an anti-IL-20RAantibody, cell, or IL-20. A “functional” IL-20RB polypeptide or fragmentthereof as defined herein is characterized by its ability to antagonizeIL-20 inflammatory, proliferative or differentiating activity, by itsability to induce or inhibit specialized cell functions, or by itsability to bind specifically to an anti-IL-20RB antibody, cell, orIL-20. As previously described herein, IL-20 is a class II cytokine andIL-20RA and IL-20RB are characterized by class II cytokine receptorstructure and domains as described herein. Thus, the present inventionfurther contemplates using fusion proteins encompassing: (a) polypeptidemolecules comprising one or more of the domains described above; and (b)functional fragments comprising one or more of these domains. The otherpolypeptide portion of the fusion protein may be contributed by anotherclass II cytokine receptor, such as IL-10R, IL-13R, IL-20RA, Crf2-4,IL-20RA2, or by a non-native and/or an unrelated secretory signalpeptide that facilitates secretion of the fusion protein.

The present invention also provides polypeptide fragments or peptidescomprising an epitope-bearing portion of an IL-20, IL-20RA or IL-20RBpolypeptide described herein. Such fragments or peptides may comprise an“immunogenic epitope,” which is a part of a protein that elicits anantibody response when the entire protein is used as an immunogen.Immunogenic epitope-bearing peptides can be identified using standardmethods (see, for example, Geysen et al., Proc. Nat'l Acad. Sci. USA81:3998 (1983)).

In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et al., Science 219:660(1983)). Accordingly, antigenic epitope-bearing peptides andpolypeptides of the present invention are useful to raise antibodiesthat bind with the polypeptides described herein. Hopp/Woodshydrophilicity profiles can be used to determine regions that have themost antigenic potential within any of SEQ ID NO:2, SEQ ID NO:11 and SEQID NO:13 (Hopp et al., Proc. Natl. Acad. Sci.78:3824-3828, 1981; Hopp,J. Immun. Meth. 88:1-18, 1986 and Triquier et al., Protein Engineering11:153-169, 1998). The profile is based on a sliding six-residue window.Buried G, S, and T residues and exposed H, Y, and W residues wereignored. In any of IL-20, IL-20RA or IL-20RB, these regions can bedetermined by one of skill in the art.

Moreover, IL-20 antigenic epitopes within SEQ ID NO:2 as predicted by aJameson-Wolf plot, e.g., using DNASTAR Protean program (DNASTAR, Inc.,Madison, Wis.) serve as preferred antigenic epitopes, and can bedetermined by one of skill in the art. Such antigenic epitopes include:amino acid residues 42 (Ile) to 102 (Asp) of SEQ ID NO:2; amino acidresidues 42 (Ile) to 60 (Ile) of SEQ ID NO:2; amino acid residues 42(Ile) to 69 (Glu) of SEQ ID NO:2; amino acid residues 42 (Ile) to 81(Cys) of SEQ ID NO:2; amino acid residues 42 (Ile) to 96 (Lys) of SEQ IDNO:2; amino acid residues 42 (Ile) to 102 (Asp) of SEQ ID NO:2; aminoacid residues 60 (Ile) to 69 (Glu) of SEQ ID NO:2; amino acid residues60 (Ile) to 81 (Cys) of SEQ ID NO:2; amino acid residues 60 (Ile) to 96(Lys) of SEQ ID NO:2; amino acid residues 60 (Ile) to 102 (Asp) of SEQID NO:2; amino acid residues 69 (Glu) to 81 (Cys) of SEQ ID NO:2; aminoacid residues 69 (Glu) to 96 (Lys) of SEQ ID NO:2; amino acid residues69 (Glu).to 102 (Asp) of SEQ ID NO:2; amino acid residues 81 (Cys) to 96(Lys) of SEQ ID NO:2; amino acid residues 81 (Cys) to 102 (Asp) of SEQID NO:2; amino acid residues 96 (Lys) to 102 (Asp) of SEQ ID NO:2.

IL-20RA antigenic epitopes include: amino acid residues 1 (Met) to 9(Leu) of SEQ ID NO:14; amino acid residues 1 (Met) to 36 (Gly) of SEQ IDNO:14; amino acid residues 1 (Met) to 41 (Ala) of SEQ ID NO:14; aminoacid residues 1 (Met) to 58 (Pro) of SEQ ID NO:14; amino acid residues 1(Met) to 63 (Gln) of SEQ ID NO:14; amino acid residues 1 (Met) to 80(Lys) of SEQ ID NO:14; amino acid residues 1 (Met) to 94 (Tyr) of SEQ IDNO:14; amino acid residues 1 (Met) to 104 (Tyr) of SEQ ID NO:14; aminoacid residues 1 (Met) to 120 (Cys) of SEQ ID NO:14; amino acid residues1 (Met) to 128 (Arg) of SEQ ID NO:14; amino acid residues 1 (Met) to 161(Trp) of SEQ ID NO:14; amino acid residues 1 (Met) to 169 (Pro) of SEQID NO:14; amino acid residues 1 (Met) to 187 (Asn) of SEQ ID NO:14;amino acid residues 1 (Met) to 194 (Trp) of SEQ ID NO:14; amino acidresidues 1 (Met) to 224(Gly) of SEQ ID NO:14; amino acid residues 1(Met) to 233 (Glu) of SEQ ID NO:14; amino acid residues 1 (Met) to 316(Ile) of SEQ ID NO:14; amino acid residues 1 (Met) to 323 (Ile) of SEQID NO:14; amino acid residues 1 (Met) to 335 (Asp) of SEQ ID NO:14;amino acid residues 1 (Met) to 340 (Asn) of SEQ ID NO:14; amino acidresidues 1 (Met) to 354 (Glu) of SEQ ID NO:14; amino acid residues 1(Met) to 371 (Cys) of SEQ ID NO:14; amino acid residues 1 (Met) to381(Ser) of SEQ ID NO:14; amino acid residues 1 (Met) to 384 (Gln) of SEQID NO:14; amino acid residues 1 (Met) to 397 (Thr) of SEQ ID NO:14;amino acid residues 1 (Met) to 412 (Ala) of SEQ ID NO:14; amino acidresidues 1 (Met) to 418 (Glu) of SEQ ID NO:14; amino acid residues 1(Met) to 462 (Gln) of SEQ ID NO:14; amino acid residues 1 (Met) to 476(Ser) of SEQ ID NO:14; amino acid residues 1 (Met) to 483 (Asp) of SEQID NO:14; amino acid residues 1 (Met) to 486 (Thr) of SEQ ID NO:14;amino acid residues 1 (Met) to 496 (Ser) of SEQ ID NO:14; amino acidresidues 1 (Met) to 511 (Gly) of SEQ ID NO:14; amino acid residues 1(Met) to 523 (Glu) of SEQ ID NO:14; amino acid residues 1 (Met) to 536(Thr) of SEQ ID NO:14; amino acid residues 36 (Gly) to 63(Gln) of SEQ IDNO:14; amino acid residues 36 (Gly) to 94 (tyr) of SEQ ID NO:14; aminoacid residues 36 (Gly) to 128 (Arg) of SEQ ID NO:14; amino acid residues36 (Gly) to 169 (Pro) of SEQ ID NO:14; amino acid residues 36 (Gly) to194 (Trp) of SEQ ID NO:14; amino acid residues 36 (Gly) to 233 (Glu) ofSEQ ID NO:14; amino acid residues 36 (Gly) to 323 (Ser) of SEQ ID NO:14;amino acid residues 36 (Gly) to 340 (Asn) of SEQ ID NO:14; amino acidresidues 36 (Gly) to 354 (Glu) of SEQ ID NO:14; amino acid residues 36(Gly) to 381 (Ser) of SEQ ID NO:14; amino acid residues 36 (Gly) to 397(Thr) of SEQ ID NO:14; amino acid residues 36 (Gly) to 418 (Glu) of SEQID NO:14; amino acid residues 36 (Gly) to 476 (Ser) of SEQ ID NO:14;amino acid residues 36 (Gly) to 486 (Thr) of SEQ ID NO:14; amino acidresidues 36 (Gly) to 511 (Gly) of SEQ ID NO:14; amino acid residues 36(Gly) to 536 (Thr) of SEQ ID NO:14; amino acid residues 58 (Pro) to 63(Gln) of SEQ ID NO:14; amino acid residues 58 (Pro) to 94 (tyr) of SEQID NO:14; amino acid residues 58 (Pro) to 128 (Arg) of SEQ ID NO:14;amino acid residues 58 (Pro) to 169 (Pro) of SEQ ID NO:14; amino acidresidues 58 (Pro) to 194 (Trp) of SEQ ID NO:14; amino acid residues 58(Pro) to 233 (Glu) of SEQ ID NO:14; amino acid residues 58 (Pro) to 323(Ser) of SEQ ID NO:14; amino acid residues 58 (Pro) to 340 (Asn) of SEQID NO:14; amino acid residues 58 (Pro) to 354 (Glu) of SEQ ID NO:14;amino acid residues 58 (Pro) to 381 (Ser) of SEQ ID NO:14; amino acidresidues 58 (Pro) to 397 (Thr) of SEQ ID NO:14; amino acid residues 58(Pro) to 418 (Glu) of SEQ ID NO:14; amino acid residues 58 (Pro) to 476(Ser) of SEQ ID NO:14; amino acid residues 58 (Pro) to 486 (Thr) of SEQID NO:14; amino acid residues 58 (Pro) to 511 (Gly) of SEQ ID NO:14;amino acid residues 58 (Pro) to 536 (Thr) of SEQ ID NO:14; amino acidresidues 80 (Lys) to 94 (tyr) of SEQ ID NO:14; amino acid residues 80(Lys) to 128 (Arg) of SEQ ID NO:14; amino acid residues 80 (Lys) to 169(Pro) of SEQ ID NO:14; amino acid residues 80 (Lys) to 194 (Trp) of SEQID NO:14; amino acid residues 80 (Lys) to 233 (Glu) of SEQ ID NO:14;amino acid residues 80 (Lys) to 323 (Ser) of SEQ ID NO:14; amino acidresidues 80 (Lys) to 340 (Asn) of SEQ ID NO:14; amino acid residues 80(Lys) to 354 (Glu) of SEQ ID NO:14; amino acid residues 80 (Lys) to 381(Ser) of SEQ ID NO:14; amino acid residues 80 (Lys) to 397 (Thr) of SEQID NO:14; amino acid residues 80 (Lys) to 418 (Glu) of SEQ ID NO:14;amino acid residues 80 (Lys) to 476 (Ser) of SEQ ID NO:14; amino acidresidues 80 (Lys) to 486 (Thr) of SEQ ID NO:14; amino acid residues 80(Lys) to 511 (Gly) of SEQ ID NO:14; amino acid residues 80 (Lys) to 536(Thr) of SEQ ID NO:14; amino acid residues 120 (Cys) to 128 (Arg) of SEQID NO:14; amino acid residues 120 (Cys) to 169 (Pro) of SEQ ID NO:14;amino acid residues 120 (Cys) to 194 (Trp) of SEQ ID NO:14; amino acidresidues 120 (Cys) to 233 (Glu) of SEQ ID NO:14; amino acid residues 120(Cys) to 323 (Ser) of SEQ ID NO:14; amino acid residues 120 (Cys) to 340(Asn) of SEQ ID NO:14; amino acid residues 120 (Cys) to 354 (Glu) of SEQID NO:14; amino acid residues 120 (Cys) to 381 (Ser) of SEQ ID NO:14;amino acid residues 120 (Cys) to 397 (Thr) of SEQ ID NO:14; amino acidresidues 120 (Cys) to 418 (Glu) of SEQ ID NO:14; amino acid residues 120(Cys) to 476 (Ser) of SEQ ID NO:14; amino acid residues 120 (Cys) to 486(Thr) of SEQ ID NO:14; amino acid residues 120 (Cys) to 511 (Gly) of SEQID NO:14; amino acid residues 120 (Cys) to 536 (Thr) of SEQ ID NO:14;amino acid residues 161 (Trp) to 169 (Pro) of SEQ ID NO:14; amino acidresidues 161 (Trp) to 194 (Trp) of SEQ ID NO:14; amino acid residues 161(Trp) to 233 (Glu) of SEQ ID NO:14; amino acid residues 161 (Trp) to 323(Ser) of SEQ ID NO:14; amino acid residues 161 (Trp) to 340 (Asn) of SEQID NO:14; amino acid residues 161 (Trp) to 354 (Glu) of SEQ ID NO:14;amino acid residues 161 (Trp) to 381 (Ser) of SEQ ID NO:14; amino acidresidues 161 (Trp) to 397 (Thr) of SEQ ID NO:14; amino acid residues 161(Trp) to 418 (Glu) of SEQ ID NO:14; amino acid residues 161 (Trp) to 476(Ser) of SEQ ID NO:14; amino acid residues 161 (Trp) to 486 (Thr) of SEQID NO:14; amino acid residues 161 (Trp) to 511 (Gly) of SEQ ID NO:14;amino acid residues 161 (Trp) to 536 (Thr) of SEQ ID NO:14; amino acidresidues 187 (Asn) to 194 (Trp) of SEQ ID NO:14; amino acid residues 187(Asn) to 233 (Glu) of SEQ ID NO:14; amino acid residues 187 (Asn) to 323(Ser) of SEQ ID NO:14; amino acid residues 187 (Asn) to 340 (Asn) of SEQID NO:14; amino acid residues 187 (Asn) to 354 (Glu) of SEQ ID NO:14;amino acid residues 187 (Asn) to 381 (Ser) of SEQ ID NO:14; amino acidresidues 187 (Asn) to 397 (Thr) of SEQ ID NO:14; amino acid residues 187(Asn) to 418 (Glu) of SEQ ID NO:14; amino acid residues 187 (Asn) to 476(Ser) of SEQ ID NO:14; amino acid residues 187 (Asn) to 486 (Thr) of SEQID NO:14; amino acid residues 187 (Asn) to 511 (Gly) of SEQ ID NO:14;amino acid residues 187 (Asn) to 536 (Thr) of SEQ ID NO:14; amino acidresidues 224 (Gly) to 233 (Glu) of SEQ ID NO:14; amino acid residues 224(Gly) to 323 (Ser) of SEQ ID NO:14; amino acid residues 224 (Gly) to 340(Asn) of SEQ ID NO:14; amino acid residues 224 (Gly) to 354 (Glu) of SEQID NO:14; amino acid residues 224 (Gly) to 381 (Ser) of SEQ ID NO:14;amino acid residues 224 (Gly) to 397 (Thr) of SEQ ID NO:14; amino acidresidues 224 (Gly) to 418 (Glu) of SEQ ID NO:14; amino acid residues 224(Gly) to 476 (Ser) of SEQ ID NO:14; amino acid residues 224 (Gly) to 486(Thr) of SEQ ID NO:14; amino acid residues 224 (Gly) to 511 (Gly) of SEQID NO:14; amino acid residues 224 (Gly) to 536 (Thr) of SEQ ID NO:14;amino acid residues 316 (Ile) to 323 (Ser) of SEQ ID NO:14; amino acidresidues 316 (Ile) to 340 (Asn) of SEQ ID NO:14; amino acid residues 316(Ile) to 354 (Glu) of SEQ ID NO:14; amino acid residues 316 (Ile) to 381(Ser) of SEQ ID NO:14; amino acid residues 316 (Ile) to 397 (Thr) of SEQID NO:14; amino acid residues 316 (Ile) to 418 (Glu) of SEQ ID NO:14;amino acid residues 316 (Ile) to 476 (Ser) of SEQ ID NO:14; amino acidresidues 316 (Ile) to 486 (Thr) of SEQ ID NO:14; amino acid residues 316(Ile) to 511 (Gly) of SEQ ID NO:14; amino acid residues 316 (Ile) to 536(Thr) of SEQ ID NO:14; amino acid residues 335 (Asp) to 340 (Asn) of SEQID NO:14; amino acid residues 335 (Asp) to 354 (Glu) of SEQ ID NO:14;amino acid residues 335 (Asp) to 381 (Ser) of SEQ ID NO:14; amino acidresidues 335 (Asp) to 397 (Thr) of SEQ ID NO:14; amino acid residues 335(Asp) to 418 (Glu) of SEQ ID NO:14; amino acid residues 335 (Asp) to 476(Ser) of SEQ ID NO:14; amino acid residues 335 (Asp) to 486 (Thr) of SEQID NO:14; amino acid residues 335 (Asp) to 511 (Gly) of SEQ ID NO:14;amino acid residues 335 (Asp) to 536 (Thr) of SEQ ID NO:14; amino acidresidues 371(Cys) to 381 (Ser) of SEQ ID NO:14; amino acid residues 371(Cys) to 397 (Thr) of SEQ ID NO:14; amino acid residues 371 (Cys) to 418(Glu) of SEQ ID NO:14; amino acid residues 371(Cys) to 476 (Ser) of SEQID NO:14; amino acid residues 371(Cys) to 486 (Thr) of SEQ ID NO:14;amino acid residues 371 (Cys) to 511 (Gly) of SEQ ID NO:14; amino acidresidues 371 (Cys) to 536 (Thr) of SEQ ID NO:14; amino acid residues 384(Gln) to 397 (Thr) of SEQ ID NO:14; amino acid residues 384 (Gln) to 418(Glu) of SEQ ID NO:14; amino acid residues 384 (Gin) to 476 (Ser) of SEQID NO:14; amino acid residues 384 (Gin) to 486 (Thr) of SEQ ID NO:14;amino acid residues 384 (Gln) to 511 (Gly) of SEQ ID NO:14; amino acidresidues 384 (Gln) to 536 (Thr) of SEQ ID NO:14; amino acid residues 412(Ala) to 418 (Glu) of SEQ ID NO:14; amino acid residues 412 (Ala) to 476(Ser) of SEQ ID NO:14; amino acid residues 412 (Ala) to 486 (Thr) of SEQID NO:14; amino acid residues 412 (Ala) to 511 (Gly) of SEQ ID NO:14;amino acid residues 412 (Ala) to 536 (Thr) of SEQ ID NO:14; amino acidresidues 462 (Gin) to 476 (Ser) of SEQ ID NO:14; amino acid residues 462(Gln) to 486 (Thr) of SEQ ID NO:14; amino acid residues 462 (Gln) to 511(Gly) of SEQ ID NO:14; amino acid residues 462 (Gin) to 536 (Thr) of SEQID NO:14; amino acid residues 483 (Asp) to 486 (Thr) of SEQ ID NO:14;amino acid residues 483 (Asp) to 511 (Gly) of SEQ ID NO:14; amino acidresidues 483(Asp) to 536 (Thr) of SEQ ID NO:14; amino acid residues 496(Ser) to 511 (Gly) of SEQ ID NO:14; amino acid residues 496 (Ser) to 536(Thr) of SEQ ID NO:14; amino acid residues 523 (Glu) to 536 (Thr) of SEQID NO:14.

IL-20RB antigenic epitopes within SEQ ID NO:21 include: amino acidresidues 70 (Tyr) to 74 (Tyr) of SEQ ID NO:21; amino acid residues 70(Tyr) to 101 (Asp) of SEQ ID NO:21; amino acid residues 70 (Tyr) to 135(Ser) of SEQ ID NO:21; amino acid residues 70 (Tyr) to 178 (Glu) of SEQID NO:21; amino acid residues 70 (Tyr) to 283 (Lys) of SEQ ID) NO:21;amino acid residues 92 (Thr) to 101 (Asp) of SEQ ID NO:21; amino acidresidues 92 (Thr) to 135 (Ser) of SEQ ID NO:21; amino acid residues 92(Thr) to 178 (Glu) of SEQ ID NO:21; amino acid residues 92 (Thr) to 283(Lys) of SEQ ID NO:21; amino acid residues 130 (Pro) to 135 (Ser) of SEQID NO:21; amino acid residues 130 (Pro) to 178 (Glu) of SEQ ID NO:21;amino acid residues 130 (Pro) to 283 (Lys) of SEQ ID NO:21; amino acidresidues 171 (Arg) to 178 (Glu) of SEQ ID NO:21; amino acid residues 171(Arg) to 283 (Lys) of SEQ ID NO:21; amino acid residues 279 (Asn) to 283(Lys) of SEQ ID NO:21.

Antigenic epitope-bearing peptides and polypeptides can contain at leastfour to ten amino acids, at least ten to fifteen amino acids, or about15 to about 30 amino acids of an amino acid sequence disclosed herein.Such epitope-bearing peptides and polypeptides can be produced byfragmenting an IL-20, IL-20RA or IL-20RB polypeptide, or by chemicalpeptide synthesis, as described herein. Moreover, epitopes can beselected by phage display of random peptide libraries (see, for example,Lane and Stephen, Curr. Opin. Immunol. 5:268 (1993), and Cortese et al.,Curr. Opin. Biotechnol. 7:616 (1996)). Standard methods for identifyingepitopes and producing antibodies from small peptides that comprise anepitope are described, for example, by Mole, “Epitope Mapping,” inMethods in Molecular Biology, Vol. 10, Manson (ed.), pages 105-116 (TheHumana Press, Inc. 1992), Price, “Production and Characterization ofSynthetic Peptide-Derived Antibodies,” in Monoclonal Antibodies:Production, Engineering, and Clinical Application, Ritter and Ladyman(eds.), pages 60-84 (Cambridge University Press 1995), and Coligan etal. (eds.), Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages9.4.1-9.4.11 (John Wiley & Sons 1997).

For any IL-20, IL-20RA or IL-20RB polypeptide, including variants andfusion proteins, one of ordinary skill in the art can readily generate afully degenerate polynucleotide sequence encoding that variant using theinformation set forth in Tables 1 and 2 above. Moreover, those of skillin the art can use standard software to devise IL-20, IL-20RA andIL-20RB variants based upon the nucleotide and amino acid sequencesdescribed herein.

5. Production of IL-20, IL-20RA and IL-20RB Polypeptides

The polypeptides of the present invention, including full-lengthpolypeptides; soluble monomeric, homodimeric, heterodimeric andmultimeric receptors; full-length receptors; receptor fragments (e.g.ligand-binding fragments and antigenic epitopes), functional fragments,and fusion proteins, can be produced in recombinant host cells followingconventional techniques. To express an IL-20, IL-20RA or IL-20RB gene, anucleic acid molecule encoding the polypeptide must be operably linkedto regulatory sequences that control transcriptional expression in anexpression vector and then, introduced into a host cell. In addition totranscriptional regulatory sequences, such as promoters and enhancers,expression vectors can include translational regulatory sequences and amarker gene which is suitable for selection of cells that carry theexpression vector.

Expression vectors that are suitable for production of a foreign proteinin eukaryotic cells typically contain (1) prokaryotic DNA elementscoding for a bacterial replication origin and an antibiotic resistancemarker to provide for the growth and selection of the expression vectorin a bacterial host; (2) eukaryotic DNA elements that control initiationof transcription, such as a promoter; and (3) DNA elements that controlthe processing of transcripts, such as a transcriptiontermination/polyadenylation sequence. As discussed above, expressionvectors can also include nucleotide sequences encoding a secretorysequence that directs the heterologous polypeptide into the secretorypathway of a host cell. For example, a IL-20RA expression vector maycomprise a IL-20RA gene and a secretory sequence derived from anysecreted gene.

The IL-20, IL-20RA and IL-20RB proteins of the present invention may beexpressed in mammalian cells. Examples of suitable mammalian host cellsinclude African green monkey kidney cells (Vero; ATCC CRL 1587), humanembryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster kidneycells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidneycells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCCCCL61; CHO DG44 (Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986)),rat pituitary cells (GHI; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rathepatoma cells (H-4-II-E; ATCC CRL 1548) SV40-transformed monkey kidneycells (COS-1; ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCCCRL 1658).

For a mammalian host, the transcriptional and translational regulatorysignals may be derived from mammalian viral sources, for example,adenovirus, bovine papilloma virus, simian virus, or the like, in whichthe regulatory signals are associated with a particular gene which has ahigh level of expression. Suitable transcriptional and translationalregulatory sequences also can be obtained from mammalian genes, forexample, actin, collagen, myosin, and metallothionein genes.

Transcriptional regulatory sequences include a promoter regionsufficient to direct the initiation of RNA synthesis. Suitableeukaryotic promoters include the promoter of the mouse metallothionein Igene (Hamer et al., J. Molec. Appl. Genet. 1:273 (1982)), the TKpromoter of Herpes virus (McKnight, Cell 31:355 (1982)), the SV40 earlypromoter (Benoist et al., Nature 290:304 (1981)), the Rous sarcoma viruspromoter (Gorman et al., Proc. Nat'l Acad. Sci. USA 79:6777 (1982)), thecytomegalovirus promoter (Foecking et al., Gene 45:101 (1980)), and themouse mammary tumor virus promoter (see, generally, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163-181 (John Wiley & Sons, Inc. 1996)).

Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNApolymerase promoter, can be used to control IL-20RA gene expression inmammalian cells if the prokaryotic promoter is regulated by a eukaryoticpromoter (Zhou et al., Mol. Cell. Biol. 10:4529 (1990), and Kaufman etal., Nucl. Acids Res. 19:4485 (1991)).

In certain embodiments, a DNA sequence encoding IL-20 or an IL-20RA orIL-20RB soluble receptor polypeptide, or a fragment of an IL-20, IL-20RAor IL-20RB polypeptide is operably linked to other genetic elementsrequired for its expression, generally including a transcriptionpromoter and terminator, within an expression vector. The vector willalso commonly contain one or more selectable markers and one or moreorigins of replication, although those skilled in the art will recognizethat within certain systems selectable markers may be provided onseparate vectors, and replication of the exogenous DNA may be providedby integration into the host cell genome. Selection of promoters,terminators, selectable markers, vectors and other elements is a matterof routine design within the level of ordinary skill in the art. Manysuch elements are described in the literature and are available throughcommercial suppliers. Multiple components of a soluble receptor complexcan be co-transfected on individual expression vectors or be containedin a single expression vector. Such techniques of expressing multiplecomponents of protein complexes are well known in the art.

An expression vector can be introduced into host cells using a varietyof standard techniques including calcium phosphate transfection,liposome-mediated transfection, microprojectile-mediated delivery,electroporation, and the like. The transfected cells can be selected andpropagated to provide recombinant host cells that comprise theexpression vector stably integrated in the host cell genome. Techniquesfor introducing vectors into eukaryotic cells and techniques forselecting such stable transformants using a dominant selectable markerare described, for example, by Ausubel (1995) and by Murray (ed.), GeneTransfer and Expression Protocols (Humana Press 1991).

For example, one suitable selectable marker is a gene that providesresistance to the antibiotic neomycin. In this case, selection iscarried out in the presence of a neomycin-type drug, such as G-418 orthe like. Selection systems can also be used to increase the expressionlevel of the gene of interest, a process referred to as “amplification.”Amplification is carried out by culturing transfectants in the presenceof a low level of the selective agent and then increasing the amount ofselective agent to select for cells that produce high levels of theproducts of the introduced genes. A suitable amplifiable selectablemarker is dihydrofolate reductase (DHFR), which confers resistance tomethotrexate. Other drug resistance genes (e.g., hygromycin resistance,multi-drug resistance, puromycin acetyltransferase) can also be used.Alternatively, markers that introduce an altered phenotype, such asgreen fluorescent protein, or cell surface proteins such as CD4, CD8,Class I MHC, placental alkaline phosphatase may be used to sorttransfected cells from untransfected cells by such means as FACS sortingor magnetic bead separation technology.

IL-20, IL-20RA or IL-20RB polypeptides can also be produced by culturedmammalian cells using a viral delivery system. Exemplary viruses forthis purpose include adenovirus, retroviruses, herpesvirus, vacciniavirus and adeno-associated virus (AAV). Adenovirus, a double-strandedDNA virus, is currently the best studied gene transfer vector fordelivery of heterologous nucleic acid (for a review, see Becker et al.,Meth. Cell Biol. 43:161 (1994), and Douglas and Curiel, Science &Medicine 4:44 (1997)). Advantages of the adenovirus system include theaccommodation of relatively large DNA inserts, the ability to grow tohigh-titer, the ability to infect a broad range of mammalian cell types,and flexibility that allows use with a large number of available vectorscontaining different promoters.

By deleting portions of the adenovirus genome, larger inserts (up to 7kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. An option is to delete theessential E1 gene from the viral vector, which results in the inabilityto replicate unless the E1 gene is provided by the host cell. Adenovirusvector-infected human 293 cells (ATCC Nos. CRL-1573, 45504, 45505), forexample, can be grown as adherent cells or in suspension culture atrelatively high cell density to produce significant amounts of protein(see Garnier et al., Cytotechnol. 15:145 (1994)).

IL-20, IL-20RA or IL-20RB can also be expressed in other highereukaryotic cells, such as avian, fungal, insect, yeast, or plant cells.The baculovirus system provides an efficient means to introduce clonedgenes into insect cells. Suitable expression vectors are based upon theAutographa californica multiple nuclear polyhedrosis virus (AcMNPV), andcontain well-known promoters such as Drosophila heat shock protein (hsp)70 promoter, Autographa californica nuclear polyhedrosis virusimmediate-early gene promoter (ie-1) and the delayed early 39K promoter,baculovirus pI0 promoter, and the Drosophila metallothionein promoter. Asecond method of making recombinant baculovirus utilizes atransposon-based system described by Luckow (Luckow, et al., J. Virol.67:4566 (1993)). This system, which utilizes transfer vectors, is soldin the BAC-to-BAC kit (Life Technologies, Rockville, Md.). This systemutilizes a transfer vector, PFASTBAC (Life Technologies) containing aTn7 transposon to move the DNA encoding the polypeptide into abaculovirus genome maintained in E. coli as a large plasmid called a“bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990),Bonning, et al., J. Gen. Virol. 75:1551 (1994), and Chazenbalk, andRapoport, J. Biol. Chem. 270:1543 (1995). In addition, transfer vectorscan include an in-frame fusion with DNA encoding an epitope tag at theC- or N-terminus of the expressed polypeptide, for example, a Glu-Gluepitope tag (Grussenmeyer et al., Proc. Nat'l Acad. Sci. 82:7952(1985)). Using a technique known in the art, a transfer vectorcontaining a gene is transformed into E. coli, and screened for bacmidswhich contain an interrupted lacZ gene indicative of recombinantbaculovirus. The bacmid DNA containing the recombinant baculovirusgenome is then isolated using common techniques.

The illustrative PFASTBAC vector can be modified to a considerabledegree. For example, the polyhedrin promoter can be removed andsubstituted with the baculovirus basic protein promoter (also known asPcor, p6.9 or MP promoter) which is expressed earlier in the baculovirusinfection, and has been shown to be advantageous for expressing secretedproteins (see, for example, Hill-Perkins and Possee, J. Gen. Virol.71:971 (1990), Bonning, et al., J. Gen. Virol. 75:1551 (1994), andChazenbalk and Rapoport, J. Biol. Chem. 270:1543 (1995). In suchtransfer vector constructs, a short or long version of the basic proteinpromoter can be used. Moreover, transfer vectors can be constructedwhich replace the native IL-20RA secretory signal sequences withsecretory signal sequences derived from insect proteins. For example, asecretory signal sequence from Ecdysteroid Glucosyltransferase (EGT),honey bee Melittin (Invitrogen Corporation; Carlsbad, Calif.), orbaculovirus gp67 (PharMingen: San Diego, Calif.) can be used inconstructs to replace the native secretory signal sequence.

The recombinant virus or bacmid is used to transfect host cells.Suitable insect host cells include cell lines derived from IPLB-Sf-21, aSpodoptera frugiperda pupal ovarian cell line, such as Sf9 (ATCC CRL1711), Sf21AE, and Sf21 (Invitrogen Corporation; San Diego, Calif.), aswell as Drosophila Schneider-2 cells, and the HIGH FIVEO cell line(Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).Commercially available serum-free media can be used to grow and tomaintain the cells. Suitable media are Sf900 II™ (Life Technologies) orESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRHBiosciences, Lenexa, Kans.) or Express FiveO™ (Life Technologies) forthe T. ni cells. When recombinant virus is used, the cells are typicallygrown up from an inoculation density of approximately 2-5×10⁵ cells to adensity of 1-2×10⁶ cells at which time a recombinant viral stock isadded at a multiplicity of infection (MOI) of 0.1 to 10, more typicallynear 3.

Established techniques for producing recombinant proteins in baculovirussystems are provided by Bailey et al., “Manipulation of BaculovirusVectors,” in Methods in Molecular Biology, Volume 7: Gene Transfer andExpression Protocols, Murray (ed.), pages 147-168 (The Humana Press,Inc. 1991), by Patel et al., “The baculovirus expression system,” in DNACloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages205-244 (Oxford University Press 1995), by Ausubel (1995) at pages 16-37to 16-57, by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995), and by Lucknow, “Insect Cell ExpressionTechnology,” in Protein Engineering: Principles and Practice, Cleland etal. (eds.), pages 183-218 (John Wiley & Sons, Inc. 1996).

Fungal cells, including yeast cells, can also be used to express thegenes described herein. Yeast species of particular interest in thisregard include Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Suitable promoters for expression in yeast includepromoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH(alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinoldehydrogenase), and the like. Many yeast cloning vectors have beendesigned and are readily available. These vectors include YIp-basedvectors, such as YIp5, YRp vectors, such as YRp17, YEp vectors such asYEp13 and YCp vectors, such as YCp19. Methods for transforming S.cerevisiae cells with exogenous DNA and producing recombinantpolypeptides therefrom are disclosed by, for example, Kawasaki, U.S.Pat. No. 4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373, Brake,U.S. Pat. No. 4,870,008, Welch et al., U.S. Pat. No. 5,037,743, andMurray et al., U.S. Pat. No. 4,845,075. Transformed cells are selectedby phenotype determined by the selectable marker, commonly drugresistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). A suitable vector system for use inSaccharomyces cerevisiae is the POT1 vector system disclosed by Kawasakiet al. (U.S. Pat. No. 4,931,373), which allows transformed cells to beselected by growth in glucose-containing media. Additional suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311, Kingsman etal., U.S. Pat. No. 4,615,974, and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446,5,063,154, 5,139,936, and 4,661,454.

Transformation systems for other yeasts, including Hansenula polymorpha,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichiaguillermondii and Candida maltosa are known in the art. See, forexample, Gleeson et al., J. Gen. Microbiol. 132:3459 (1986), and Cregg,U.S. Pat. No. 4,882,279. Aspergillus cells may be utilized according tothe methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods fortransforming Acremonium chrysogenum are disclosed by Sumino et al., U.S.Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed byLambowitz, U.S. Pat. No. 4,486,533.

For example, the use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed by Raymond, U.S. Pat. No. 5,716,808,Raymond, U.S. Pat. No. 5,736,383, Raymond et al., Yeast 14:11-23 (1998),and in international publication Nos. WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, the promoter and terminator inthe plasmid can be that of a P. methanolica gene, such as a P.methanolica alcohol utilization gene (AUG1 or AUG2). Other usefulpromoters include those of the dihydroxyacetone synthase (DHAS), formatedehydrogenase (FMD), and catalase (CAT) genes. To facilitate integrationof the DNA into the host chromosome, it is preferred to have the entireexpression segment of the plasmid flanked at both ends by host DNAsequences. A suitable selectable marker for use in Pichia methanolica isa P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), andwhich allows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, host cells can be used in which both methanolutilization genes (AUG1 and AUG2) are deleted. For production ofsecreted proteins, host cells can be deficient in vacuolar proteasegenes (PEP4 and PRBI). Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. P. methanolica cells can betransformed by electroporation using an exponentially decaying, pulsedelectric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Expression vectors can also be introduced into plant protoplasts, intactplant tissues, or isolated plant cells. Methods for introducingexpression vectors into plant tissue include the direct infection orco-cultivation of plant tissue with Agrobacterium tumefaciens,microprojectile-mediated delivery, DNA injection, electroporation, andthe like. See, for example, Horsch et al., Science 227:1229 (1985),Klein et al., Biotechnology 10:268 (1992), and Miki et al., “Proceduresfor Introducing Foreign DNA into Plants,” in Methods in Plant MolecularBiology and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,1993).

Alternatively, IL-20, IL-20RA or IL-20RB genes can be expressed inprokaryotic host cells. Suitable promoters that can be used to expressIL-20, IL-20RA or IL-20RB polypeptides in a prokaryotic host arewell-known to those of skill in the art and include promoters capable ofrecognizing the T4, T3, Sp6 and T7 polymerases, the P_(R) and P_(L)promoters of bacteriophage lambda, the trp, recA, heat shock, lacUV5,tac, lpp-lacSpr, phoA, and lacZ promoters of E. coli, promoters of B.subtilis, the promoters of the bacteriophages of Bacillus, Streptomycespromoters, the int promoter of bacterio-phage lambda, the bla promoterof pBR322, and the CAT promoter of the chloram-phenicol acetyltransferase gene. Prokaryotic promoters have been reviewed by Glick, J.Ind. Microbiol. 1:277 (1987), Watson et al., Molecular Biology of theGene, 4th Ed. (Benjamin Cummins 1987), and by Ausubel et al. (1995).

Suitable prokaryotic hosts include E. coli and Bacillus subtilus.Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS,BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF, DH5IMCR, DH10B, DH10B/p3,DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089,CSH18, ER1451, and ER1647 (see, for example, Brown (ed.), MolecularBiology Labfax (Academic Press 1991)). Suitable strains of Bacillussubtilus include BR151, YB886, MI119, MI120, and B170 (see, for example,Hardy, “Bacillus Cloning Methods,” in DNA Cloning: A Practical Approach,Glover (ed.) (IRL Press 1985)).

When expressing a IL-20, IL-20RA or IL-20RB polypeptide in bacteria suchas E. coli, the polypeptide may be retained in the cytoplasm, typicallyas insoluble granules, or may be directed to the periplasmic space by abacterial secretion sequence. In the former case, the cells are lysed,and the granules are recovered and denatured using, for example,guanidine isothiocyanate or urea. The denatured polypeptide can then berefolded and dimerized by diluting the denaturant, such as by dialysisagainst a solution of urea and a combination of reduced and oxidizedglutathione, followed by dialysis against a buffered saline solution. Inthe latter case, the polypeptide can be recovered from the periplasmicspace in a soluble and functional form by disrupting the cells (by, forexample, sonication or osmotic shock) to release the contents of theperiplasmic space and recovering the protein, thereby obviating the needfor denaturation and refolding.

Methods for expressing proteins in prokaryotic hosts are well-known tothose of skill in the art (see, for example, Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995), Ward et al., “Genetic Manipulation andExpression of Antibodies,” in Monoclonal Antibodies: Principles andApplications, page 137 (Wiley-Liss, Inc. 1995), and Georgiou,“Expression of Proteins in Bacteria,” in Protein Engineering: Principlesand Practice, Cleland et al. (eds.), page 101 (John Wiley & Sons, Inc.1996)).

Standard methods for introducing expression vectors into bacterial,yeast, insect, and plant cells are provided, for example, by Ausubel(1995).

General methods for expressing and recovering foreign protein producedby a mammalian cell system are provided by, for example, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163 (Wiley-Liss, Inc. 1996). Standard techniques for recoveringprotein produced by a bacterial system is provided by, for example,Grisshammer et al., “Purification of over-produced proteins from E. colicells,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.(eds.), pages 59-92 (Oxford University Press 1995). Established methodsfor isolating recombinant proteins from a baculovirus system aredescribed by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995).

As an alternative, polypeptides of the present invention can besynthesized by exclusive solid phase synthesis, partial solid phasemethods, fragment condensation or classical solution synthesis. Thesesynthesis methods are well-known to those of skill in the art (see, forexample, Merrifield, J. Am. Chem. Soc. 85:2149 (1963), Stewart et al.,“Solid Phase Peptide Synthesis” (2nd Edition), (Pierce Chemical Co.1984), Bayer and Rapp, Chem. Pept. Prot. 3:3 (1986), Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach (IRL Press 1989),Fields and Colowick, “Solid-Phase Peptide Synthesis,” Methods inEnzymology Volume 289 (Academic Press 1997), and Lloyd-Williams et al.,Chemical Approaches to the Synthesis of Peptides and Proteins (CRCPress, Inc. 1997)). Variations in total chemical synthesis strategies,such as “native chemical ligation” and “expressed protein ligation” arealso standard (see, for example, Dawson et al., Science 266:776 (1994),Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997), Dawson,Methods Enzymol. 287: 34 (1997), Muir et al, Proc. Nat'l Acad. Sci. USA95:6705 (1998), and Severinov and Muir, J. Biol. Chem. 273:16205(1998)).

Peptides and polypeptides of the present invention comprise at leastsix, at least nine, or at least 15 contiguous amino acid residues of SEQID NO:2, SEQ ID NO:11 or SEQ ID NO:13. As an illustration, polypeptidescan comprise at least six, at least nine, or at least 15 contiguousamino acid residues of SEQ ID NO:2, SEQ ID NO:11 or SEQ ID NO:13. Withincertain embodiments of the invention, the polypeptides comprise 20, 30,40, 50, 100, or more contiguous residues of these amino acid sequences.Nucleic acid molecules encoding such peptides and polypeptides areuseful as polymerase chain reaction primers and probes.

Moreover, IL-20, IL-20RA and IL-20RB polypeptides and fragments thereofcan be expressed as monomers, homodimers, heterodimers, or multimerswithin higher eukaryotic cells. Such cells can be used to produce IL-20,or IL-20RA and IL-20RB monomeric, homodimeric, heterodimeric andmultimeric receptor polypeptides that comprise either at least oneIL-20RA polypeptide (“IL-20RA-comprising receptors” or“IL-20RA-comprising receptor polypeptides”), or at least one IL-20RBpolypeptide (“IL-20RB-comprising receptors” or “IL-20RB-comprisingreceptor polypeptides”); or can be used as assay cells in screeningsystems. Within one aspect of the present invention, a polypeptide ofthe present invention comprising either the IL-20RA or IL-20RBextracellular domain or both the L-20RA and IL-20RB extracellulardomains is produced by a cultured cell, and the cell is used to screenfor ligands for the receptor, including the natural ligand, IL-20, aswell as agonists and antagonists of the natural ligand. To summarizethis approach, a cDNA or gene encoding the receptor is combined withother genetic elements required for its expression (e.g., atranscription promoter), and the resulting expression vector is insertedinto a host cell. Cells that express the DNA and produce functionalreceptor are selected and used within a variety of screening systems.Each component of the monomeric, homodimeric, heterodimeric andmultimeric receptor complex can be expressed in the same cell. Moreover,the components of the monomeric, homodimeric, heterodimeric andmultimeric receptor complex can also be fused to a transmembrane domainor other membrane fusion moiety to allow complex assembly and screeningof transfectants as described above.

To assay the IL-20 antagonist polypeptides and antibodies of the presentinvention, mammalian cells suitable for use in expressing IL-20 or IL-20receptors (e.g., cells expressing IL-20RA, IL-20RB, or IL-20RA/IL-20RB)and transducing a receptor-mediated signal include cells that expressother receptor subunits that may form a functional complex with IL-20RAor IL-20RB. These subunits may include those of the interferon receptorfamily or of other class II or class I cytokine receptors, e.g., CRF2-4(Genbank Accession No. Z17227), IL-10R (Genbank Accession No.s U00672and NM_(—)001558), IL-20RA (commonly owned U.S. Pat. No. 5,965,704),zcytor7 (IL-20RA) (commonly owned U.S. Pat. No. 5,945,511),IL-20RA/IL-20RB (WIPO Publication No. WO 01/46232), and IL-9R. It isalso preferred to use a cell from the same species as the receptor to beexpressed. Within a preferred embodiment, the cell is dependent upon anexogenously supplied hematopoietic growth factor for its proliferation.Preferred cell lines of this type are the human TF-1 cell line (ATCCnumber CRL-2003) and the AML-193 cell line (ATCC number CRL-9589), whichare GM-CSF-dependent human leukemic cell lines and BaF3 (Palacios andSteinmetz, Cell 41: 727-734, (1985)) which is an IL-3 dependent murinepre-B cell line. Other cell lines include BHK, COS-1 and CHO cells.Suitable host cells can be engineered to produce the necessary receptorsubunits or other cellular component needed for the desired cellularresponse. This approach is advantageous because cell lines can beengineered to express receptor subunits from any species, therebyovercoming potential limitations arising from species specificity.Species orthologs of the human receptor cDNA can be cloned and usedwithin cell lines from the same species, such as a mouse cDNA in theBaF3 cell line. Cell lines that are dependent upon one hematopoieticgrowth factor, such as GM-CSF or IL-3, can thus be engineered to becomedependent upon another cytokine that acts through the IL-20RA receptor,such as IL-20.

Cells expressing functional receptor are used within screening assays. Avariety of suitable assays are known in the art. These assays are basedon the detection of a biological response in a target cell. One suchassay is a cell proliferation assay. Cells are cultured in the presenceor absence of a test compound, and cell proliferation is detected by,for example, measuring incorporation of tritiated thymidine or bycolorimetric assay based on the metabolic breakdown of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)(Mosman, J. Immunol. Meth. 65: 55-63, (1983)). An alternative assayformat uses cells that are further engineered to express a reportergene. The reporter gene is linked to a promoter element that isresponsive to the receptor-linked pathway, and the assay detectsactivation of transcription of the reporter gene. A preferred promoterelement in this regard is a serum response element, or SRE. See, e.g.,Shaw et al., Cell 56:563-572, (1989). A preferred such reporter gene isa luciferase gene (de Wet et al., Mol. Cell. Biol. 7:725, (1987)).Expression of the luciferase gene is detected by luminescence usingmethods known in the art (e.g., Baumgartner et al., J. Biol. Chem.269:29094-29101, (1994); Schenborn and Goiffin, Promega Notes 41:11,1993). Luciferase activity assay kits are commercially available from,for example, Promega Corp., Madison, Wis. Target cell lines of this typecan be used to screen libraries of chemicals, cell-conditioned culturemedia, fungal broths, soil samples, water samples, and the like. Forexample, a bank of cell-conditioned media samples can be assayed on atarget cell to identify cells that produce ligand. Positive cells arethen used to produce a cDNA library in a mammalian expression vector,which is divided into pools, transfected into host cells, and expressed.Media samples from the transfected cells are then assayed, withsubsequent division of pools, re-transfection, subculturing, andre-assay of positive cells to isolate a cloned cDNA encoding the ligand.

Several IL-20 responsive cell lines are known in the art or can beconstructed, for example, the Baf3/cytoR11/DIRS1 or Baf3/cytoR7/DIRS1(WIPO Publication No. WO 02/072607). Moreover several IL-20 responsivecell lines are known (Dumontier et al., J. Immunol. 164:1814-1819, 2000;Dumoutier, L. et al., Proc. Nat'l. Acad. Sci. 97:10144-10149, 2000; XieM H et al., J. Biol. Chem. 275: 31335-31339, 2000; Kotenko S V et al.,J. Biol. Chem. 276:2725-2732, 2001), as well as those that express theIL-20 receptor subunit IL-20RA and IL-20RB. For example, the followingcells are responsive to IL-20: intestinal epithelial cells HT-29(Dumoutier et al., J. Immunol. 167: 3545-3549, 2001) and Colo205, lungcancer cell line A549 and endothelial cell HUVEC (human umbilical veinendothelial cell) (Ramesh et al., Cancer Research 63: 5105-5113), andkeratinocyte cell line HaCaT (Blumberg et al., Cell 104: 9-19). Thesecells can be used in assays to assess the functionality of anti-IL-20RAor IL-20RB mAb as an IL-20 antagonist or anti-inflammatory factor.

6. Production of IL-20RA and IL-20RB Fusion Proteins and Conjugates

One general class of IL-20RA and IL-20RB analogs are variants having anamino acid sequence that is a mutation of the amino acid sequencedisclosed herein. Another general class of IL-20RA and IL-20RB analogsis provided by anti-idiotype antibodies, and fragments thereof, asdescribed below. Moreover, recombinant antibodies comprisinganti-idiotype variable domains can be used as analogs (see, for example,Monfardini et al., Proc. Assoc. Am. Physicians 108:420 (1996)). Sincethe variable domains of anti-idiotype IL-20RA and IL-20RB antibodiesmimic IL-20RA and IL-20RB, these domains can provide IL-20RA and IL-20RBbinding activity. Methods of producing anti-idiotypic catalyticantibodies are known to those of skill in the art (see, for example,Joron et al., Ann. N Y Acad. Sci. 672:216 (1992), Friboulet et al.,Appl. Biochem. Biotechnol. 47:229 (1994), and Avalle et al., Ann. N YAcad. Sci. 864:118 (1998)).

Another approach to identifying IL-20RA and IL-20RB analogs is providedby the use of combinatorial libraries. Methods for constructing andscreening phage display and other combinatorial libraries are provided,for example, by Kay et al., Phage Display of Peptides and Proteins(Academic Press 1996), Verdine, U.S. Pat. No. 5,783,384, Kay, et. al.,U.S. Pat. No. 5,747,334, and Kauffman et al., U.S. Pat. No. 5,723,323.

IL-20RA and IL-20RB polypeptides have both in vivo and in vitro uses. Asan illustration, a soluble form of IL-20RA or IL-20RB can be added tocell culture medium to inhibit the effects of IL-20 produced by thecultured cells.

Fusion proteins of IL-20RA and 1IL-20RB can be used to express 1L-20RAand IL-20RB in a recombinant host, and to isolate the produced IL-20RAor IL-20RB. As described below, particular IL-20RA or IL-20RB fusionproteins also have uses in diagnosis and therapy. One type of fusionprotein comprises a peptide that guides, for example, a IL-20RApolypeptide from a recombinant host cell. To direct a IL-20RApolypeptide into the secretory pathway of a eukaryotic host cell, asecretory signal sequence (also known as a signal peptide, a leadersequence, prepro sequence or pre sequence) is provided in the IL-20RAexpression vector. While the secretory signal sequence may be derivedfrom HL-20RA, a suitable signal sequence may also be derived fromanother secreted protein or synthesized de novo. The secretory signalsequence is operably linked to a IL-20RA-encoding sequence such that thetwo sequences are joined in the correct reading frame and positioned todirect the newly synthesized polypeptide into the secretory pathway ofthe host cell. Secretory signal sequences are commonly positioned 5′ tothe nucleotide sequence encoding the polypeptide of interest, althoughcertain secretory signal sequences may be positioned elsewhere in thenucleotide sequence of interest (see, e.g., Welch et al., U.S. Pat. No.5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

Although the secretory signal sequence of IL-20RA or IL-20RB or anotherprotein produced by mammalian cells (e.g., tissue-type plasminogenactivator signal sequence, as described, for example, in U.S. Pat. No.5,641,655) is useful for expression of IL-20RA or IL-20RB in recombinantmammalian hosts, a yeast signal sequence is preferred for expression inyeast cells. Examples of suitable yeast signal sequences are thosederived from yeast mating phermone α-factor (encoded by the MFα1 gene),invertase (encoded by the SUC2 gene), or acid phosphatase (encoded bythe PHO5 gene). See, for example, Romanos et al., “Expression of ClonedGenes in Yeast,” in DNA Cloning 2: A Practical Approach, 2^(nd) Edition,Glover and Hames (eds.), pages 123-167 (Oxford University Press 1995).

IL-20RA or IL-20RB soluble receptor polypeptides can be prepared byexpressing a truncated DNA encoding the extracellular domain, forexample, a polypeptide which contains SEQ ID NO:11 or 13, or thecorresponding region of a non-human receptor. It is preferred that theextracellular domain polypeptides be prepared in a form substantiallyfree of transmembrane and intracellular polypeptide segments. To directthe export of the receptor domain from the host cell, the receptor DNAis linked to a second DNA segment encoding a secretory peptide, such asa t-PA secretory peptide. To facilitate purification of the secretedreceptor domain, a C-terminal extension, such as a poly-histidine tag,substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-1210,(1988); available from Eastman Kodak Co., New Haven, Conn.) or anotherpolypeptide or protein for which an antibody or other specific bindingagent is available, can be fused to the receptor polypeptide. Moreover,IL-20RA antigenic epitopes from the extracellular cytokine bindingdomains are a also prepared as described above.

In an alternative approach, a receptor extracellular domain of IL-20RA,IL-20RB or other class I or II cytokine receptor component can beexpressed as a fusion with immunoglobulin heavy chain constant regions,typically an F_(c) fragment, which contains two constant region domainsand a hinge region but lacks the variable region (See, Sledziewski, A Zet al., U.S. Pat. Nos. 6,018,026 and 5,750,375). The soluble IL-20RA orIL-20RB polypeptides of the present invention include such fusions. Suchfusions are typically secreted as multimeric molecules wherein the Fcportions are disulfide bonded to each other and two receptorpolypeptides are arrayed in closed proximity to each other. Fusions ofthis type can be used to affinity purify the cognate ligand fromsolution, as an in vitro assay tool, to block signals in vitro byspecifically titrating out ligand, and as antagonists in vivo byadministering them parenterally to bind circulating ligand and clear itfrom the circulation. For Example, to purify ligand, a IL-20RA-Igchimera is added to a sample containing the ligand (e.g.,cell-conditioned culture media or tissue extracts) under conditions thatfacilitate receptor-ligand binding (typically near-physiologicaltemperature, pH, and ionic strength). The chimera-ligand complex is thenseparated by the mixture using protein A, which is immobilized on asolid support (e.g., insoluble resin beads). The ligand is then elutedusing conventional chemical techniques, such as with a salt or pHgradient. In the alternative, the chimera itself can be bound to a solidsupport, with binding and elution carried out as above. The chimeras maybe used in vivo to regulate inflammatory responses including acute phaseresponses such as serum amyloid A (SAA), C-reactive protein (CRP), andthe like. Chimeras with high binding affinity are administeredparenterally (e.g., by intramuscular, subcutaneous or intravenousinjection). Circulating molecules bind ligand and are cleared fromcirculation by normal physiological processes. For use in assays, thechimeras are bound to a support via the F_(c) region and used in anELISA format.

To assist in isolating anti-IL-20, anti-IL-20RA or anti-IL-20RB andbinding partners of the present invention, an assay system that uses aligand-binding receptor (or an antibody, one member of acomplement/anti-complement pair) or a binding fragment thereof, and acommercially available biosensor instrument (BIAcore, PharmaciaBiosensor, Piscataway, N.J.) may be advantageously employed. Suchreceptor, antibody, member of a complement/anti-complement pair orfragment is immobilized onto the surface of a receptor chip. Use of thisinstrument is disclosed by Karlsson, J. Immunol. Methods 145:229-40,1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993. Areceptor, antibody, member or fragment is covalently attached, usingamine or sulfhydryl chemistry, to dextran fibers that are attached togold film within the flow cell. A test sample is passed through thecell. If a ligand, epitope, or opposite member of thecomplement/anti-complement pair is present in the sample, it will bindto the immobilized receptor, antibody or member, respectively, causing achange in the refractive index of the medium, which is detected as achange in surface plasmon resonance of the gold film. This system allowsthe determination of on- and off-rates, from which binding affinity canbe calculated, and assessment of stoichiometry of binding.Alternatively, ligand/receptor binding can be analyzed using SELDI(TM)technology (Ciphergen, Inc., Palo Alto, Calif.). Moreover, BIACOREtechnology, described above, can be used to be used in competitionexperiments to determine if different momnoclonal antibodies bind thesame or different epitopes on IL-20, IL-20RA and IL-20RB polypeptides,and as such, be used to aid in epitope mapping of neutralizingantibodies of the present invention that bind, or antagonize IL-20.

Ligand-binding receptor polypeptides can also be used within other assaysystems known in the art. Such systems include Scatchard analysis fordetermination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51:660-72, 1949) and calorimetric assays (Cunningham et al., Science253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

The present invention further provides a variety of other polypeptidefusions and related multimeric proteins comprising one or morepolypeptide fusions. For example, a soluble IL-20RA or IL-20RB receptorcan be prepared as a fusion to a dimerizing protein as disclosed in U.S.Pat. Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in thisregard include immunoglobulin constant region domains, e.g., IgGγ1, andthe human κ light chain. Immunoglobulin-soluble IL-20RA or IL-20RBfusions can be expressed in genetically engineered cells to produce avariety of multimeric IL-20RA or IL-20RB receptor analogs. Auxiliarydomains can be fused to soluble IL-20RA or IL-20RB receptor to targetthem to specific cells, tissues, or macromolecules (e.g., collagen, orcells expressing the IL-20RA and IL-20RB ligands, or IL-20). A IL-20RAor IL-20RB polypeptide can be fused to two or more moieties, such as anaffinity tag for purification and a targeting domain. Polypeptidefusions can also comprise one or more cleavage sites, particularlybetween domains. See, Tuan et al., Connective Tissue Research 34:1-9,1996.

In bacterial cells, it is often desirable to express a heterologousprotein as a fusion protein to decrease toxicity, increase stability,and to enhance recovery of the expressed protein. For example, any ofthe polypeptides IL-20, IL-20RA or IL-20RB can be expressed as a fusionprotein comprising a glutathione S-transferase polypeptide. GlutathioneS-transferease fusion proteins are typically soluble, and easilypurifiable from E. coli lysates on immobilized glutathione columns. Insimilar approaches, a fusion protein comprising a maltose bindingprotein polypeptide can be isolated with an amylose resin column, whilea fusion protein comprising the C-terminal end of a truncated Protein Agene can be purified using IgG-Sepharose. Established techniques forexpressing a heterologous polypeptide as a fusion protein in a bacterialcell are described, for example, by Williams et al., “Expression ofForeign Proteins in E. coli Using Plasmid Vectors and Purification ofSpecific Polyclonal Antibodies,” in DNA Cloning 2: A Practical Approach,2^(nd) Edition, Glover and Hames (Eds.), pages 15-58 (Oxford UniversityPress 1995). In addition, commercially available expression systems areavailable. For example, the PINPOINT Xa protein purification system(Promega Corporation; Madison, Wis.) provides a method for isolating afusion protein comprising a polypeptide that becomes biotinylated duringexpression with a resin that comprises avidin.

Peptide tags that are useful for isolating heterologous polypeptidesexpressed by either prokaryotic or eukaryotic cells includepolyHistidine tags (which have an affinity for nickel-chelating resin),c-myc tags, calmodulin binding protein (isolated with calmodulinaffinity chromatography), substance P, the RYIRS tag (which binds withanti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which bindswith anti-FLAG antibodies). See, for example, Luo et al., Arch. Biochem.Biophys. 329:215 (1996), Morganti et al., Biotechnol. Appl. Biochem.23:67 (1996), and Zheng et al., Gene 186:55 (1997). Nucleic acidmolecules encoding such peptide tags are available, for example, fromSigma-Aldrich Corporation (St. Louis, Mo.).

Another form of fusion protein comprises either an IL-20, an IL-20RA ora IL-20RB polypeptide and an immunoglobulin heavy chain constant region,typically an F_(c) fragment, which contains two or three constant regiondomains and a hinge region but lacks the variable region. As anillustration, Chang et al., U.S. Pat. No. 5,723,125, describe a fusionprotein comprising a human interferon and a human immunoglobulin Fcfragment. The C-terminal of the interferon is linked to the N-terminalof the Fc fragment by a peptide linker moiety. An example of a peptidelinker is a peptide comprising primarily a T cell inert sequence, whichis immunologically inert. An exemplary peptide linker has the amino acidsequence: GGSGG SGGGG SGGGG S (SEQ ID NO:14).

In another variation, a IL-20, IL-20RA or IL-20RB fusion proteincomprises an IgG sequence, a IL-20, IL-20RA or IL-20RB moiety covalentlyjoined to the aminoterminal end of the IgG sequence, and a signalpeptide that is covalently joined to the aminoterminal of the IL-20,IL-20RA or IL-20RB moiety, and wherein the IgG sequence consists of thefollowing elements in the following order: a hinge region, a CH₂ domain,and a CH₃ domain. Accordingly, the IgG sequence lacks a CH₁ domain. TheIL-20, IL-20RA or IL-20RB moiety displays its respective activity, asdescribed herein, such as the ability to bind with IL-20 (IL-20RA andIL-20RB) or its ability to bind to its respective receptor (IL-20). Thisgeneral approach to producing fusion proteins that comprise bothantibody and nonantibody portions has been described by LaRochelle etal., EP 742830 (WO 95/21258).

Fusion proteins comprising a IL-20, IL-20RA or IL-20RB moiety and an Fcmoiety can be used, for example, as an in vitro assay tool. For example,the presence of a L-20RA ligand (i.e. IL-20) in a biological sample canbe detected using a IL-20RA-immunoglobulin fusion protein, in which theIL-20RA moiety is used to bind the ligand, and a macromolecule, such asProtein A or anti-Fc antibody, is used to bind the fusion protein to asolid support. Such systems can be used to identify agonists andantagonists that interfere with the binding of a IL-20RA ligands, e.g.,IL-20, to their receptor.

Other examples of antibody fusion proteins include polypeptides thatcomprise an antigen-binding domain and a IL-20RA or IL-20RB fragmentthat contains an extracellular domain. Such molecules can be used totarget particular tissues for the benefit of binding activity.

The present invention further provides a variety of other polypeptidefusions. For example, part or all of a domain(s) conferring a biologicalfunction can be swapped between any of IL-20, IL-20RA or IL-20RB withthe functionally equivalent domain(s) from another member of thecytokine receptor family. Polypeptide fusions can be expressed inrecombinant host cells to produce a variety of IL-20, IL-20RA or IL-20RBfusion analogs. A IL-20, IL-20RA or IL-20RB polypeptide can be fused totwo or more moieties or domains, such as an affinity tag forpurification and a targeting domain. Polypeptide fusions can alsocomprise one or more cleavage sites, particularly between domains. See,for example, Tuan et al., Connective Tissue Research 34:1 (1996).

Fusion proteins can be prepared by methods known to those skilled in theart by preparing each component of the fusion protein and chemicallyconjugating them. Alternatively, a polynucleotide encoding bothcomponents of the fusion protein in the proper reading frame can begenerated using known techniques and expressed by the methods describedherein. General methods for enzymatic and chemical cleavage of fusionproteins are described, for example, by Ausubel (1995) at pages 16-19 to16-25.

IL-20, IL-20RA or IL-20RB binding domains can be further characterizedby physical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids of IL-20RA ligand agonists. See, for example, de Vos etal., Science 255:306 (1992), Smith et al., J. Mol. Biol. 224:899 (1992),and Wlodaver et al., FEBS Lett. 309:59 (1992).

The present invention also contemplates chemically modified IL-20,IL-20RA or IL-20RB compositions or conjugates, in which an IL-20,IL-20RA or IL-20RB polypeptide is linked with a polymer. Typically, thepolymer is water soluble so that the conjugate does not precipitate inan aqueous environment, such as a physiological environment. An exampleof a suitable polymer is one that has been modified to have a singlereactive group, such as an active ester for acylation, or an aldehydefor alkylation. In this way, the degree of polymerization can becontrolled. An example of a reactive aldehyde is polyethylene glycolpropionaldehyde, or mono-(C1-C10) alkoxy, or aryloxy derivatives thereof(see, for example, Harris, et al., U.S. Pat. No. 5,252,714). The polymermay be branched or unbranched. Moreover, a mixture of polymers can beused to produce conjugates.

IL-20, IL-20RA or IL-20RB conjugates used for therapy can comprisepharmaceutically acceptable water-soluble polymer moieties. Suitablewater-soluble polymers include polyethylene glycol (PEG),monomethoxy-PEG, mono-(C1-C10)alkoxy-PEG, aryloxy-PEG, poly-(N-vinylpyrrolidone)PEG, tresyl monomethoxy PEG, PEG propionaldehyde,bis-succinimidyl carbonate PEG, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or othercarbohydrate-based polymers. Suitable PEG may have a molecular weightfrom about 600 to about 60,000, including, for example, 5,000, 12,000,20,000 and 25,000. A IL-20, IL-20RA or IL-20RB conjugate can alsocomprise a mixture of such water-soluble polymers.

One example of a IL-20, IL-20RA or IL-20RB conjugate comprises a IL-20,IL-20RA or IL-20RB moiety and a polyalkyl oxide moiety attached to theN-terminus of the IL-20, IL-20RA or IL-20RB moiety. PEG is one suitablepolyalkyl oxide. As an illustration, IL-20RA can be modified with PEG, aprocess known as “PEGylation.” PEGylation of IL-20, IL-20RA or IL-20RBcan be carried out by any of the PEGylation reactions known in the art(see, for example, EP 0 154 316, Delgado et al., Critical Reviews inTherapeutic Drug Carrier Systems 9:249 (1992), Duncan and Spreafico,Clin. Pharmacokinet. 27:290 (1994), and Francis et al., Int J Hematol68:1 (1998)). For example, PEGylation can be performed by an acylationreaction or by an alkylation reaction with a reactive polyethyleneglycol molecule. In an alternative approach, IL-20RA conjugates areformed by condensing activated PEG, in which a terminal hydroxy or aminogroup of PEG has been replaced by an activated linker (see, for example,Karasiewicz et al., U.S. Pat. No. 5,382,657).

PEGylation by acylation typically requires reacting an active esterderivative of PEG with a IL-20, IL-20RA or IL-20RB polypeptide. Anexample of an activated PEG ester is PEG esterified toN-hydroxysuccinimide. As used herein, the term “acylation” includes thefollowing types of linkages between IL-20, IL-20RA or IL-20RB and awater soluble polymer: amide, carbamate, urethane, and the like. Methodsfor preparing PEGylated IL-20, IL-20RA or IL-20RB by acylation willtypically comprise the steps of (a) reacting a IL-20, IL-20RA or IL-20RBpolypeptide with PEG (such as a reactive ester of an aldehyde derivativeof PEG) under conditions whereby one or more PEG groups attach to IL-20,IL-20RA or IL-20RB, and (b) obtaining the reaction product(s).Generally, the optimal reaction conditions for acylation reactions willbe determined based upon known parameters and desired results. Forexample, the larger the ratio of, for instance, PEG:IL-20RA, the greaterthe percentage of polyPEGylated IL-20RA product.

The product of PEGylation by acylation is typically a polyPEGylatedIL-20, IL-20RA or IL-20RB product, wherein the lysine ε-amino groups arePEGylated via an acyl linking group. An example of a connecting linkageis an amide. Typically, the resulting IL-20, IL-20RA or IL-20RB will beat least 95% mono-, di-, or tri-pegylated, although some species withhigher degrees of PEGylation may be formed depending upon the reactionconditions. PEGylated species can be separated from unconjugated IL-20,IL-20RA or IL-20RB polypeptides using standard purification methods,such as dialysis, ultrafiltration, ion exchange chromatography, affinitychromatography, and the like.

PEGylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with IL-20, IL-20RA or IL-20RB in the presence of areducing agent. PEG groups can be attached to the polypeptide via a—CH₂—NH group.

Derivatization via reductive alkylation to produce a monoPEGylatedproduct takes advantage of the differential reactivity of differenttypes of primary amino groups available for derivatization. Typically,the reaction is performed at a pH that allows one to take advantage ofthe pKa differences between the ε-amino groups of the lysine residuesand the α-amino group of the N-terminal residue of the protein. By suchselective derivatization, attachment of a water-soluble polymer thatcontains a reactive group such as an aldehyde, to a protein iscontrolled. The conjugation with the polymer occurs predominantly at theN-terminus of the protein without significant modification of otherreactive groups such as the lysine side chain amino groups. The presentinvention provides a substantially homogenous preparation of IL-20,IL-20RA or IL-20RB monopolymer conjugates.

Reductive alkylation to produce a substantially homogenous population ofmonopolymer IL-20, IL-20RA or IL-20RB conjugate molecule can comprisethe steps of: (a) reacting a IL-20, IL-20RA or IL-20RB polypeptide witha reactive PEG under reductive alkylation conditions at a pH suitable topermit selective modification of the ax-amino group at the aminoterminus of the IL-20, IL-20RA or IL-20RB, and (b) obtaining thereaction product(s). The reducing agent used for reductive alkylationshould be stable in aqueous solution and able to reduce only the Schiffbase formed in the initial process of reductive alkylation. Illustrativereducing agents include sodium borohydride, sodium cyanoborohydride,dimethylamine borane, trimethylamine borane, and pyridine borane.

For a substantially homogenous population of monopolymer IL-20, IL-20RAor IL-20RB conjugates, the reductive alkylation reaction conditions arethose that permit the selective attachment of the water-soluble polymermoiety to the N-terminus of IL-20, IL-20RA or IL-20RB. Such reactionconditions generally provide for pKa differences between the lysineamino groups and the ax-amino group at the N-terminus. The pH alsoaffects the ratio of polymer to protein to be used. In general, if thepH is lower, a larger excess of polymer to protein will be desiredbecause the less reactive the N-terminal ax-group, the more polymer isneeded to achieve optimal conditions. If the pH is higher, the polymer:IL-20, IL-20RA or IL-20RB need not be as large because more reactivegroups are available. Typically, the pH will fall within the range of 3to 9, or 3 to 6. This method can be employed for making IL-20, IL-20RAor IL-20RB-comprising homodimeric, heterodimeric or multimeric solublereceptor conjugates.

Another factor to consider is the molecular weight of the water-solublepolymer. Generally, the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.For PEGylation reactions, the typical molecular weight is about 2 kDa toabout 100 kDa, about 5 kDa to about 50 kDa, or about 12 kDa to about 25kDa. The molar ratio of water-soluble polymer to IL-20, IL-20RA orIL-20RB will generally be in the range of 1:1 to 100:1. Typically, themolar ratio of water-soluble polymer to IL-20, IL-20RA or IL-20RB willbe 1:1 to 20:1 for polyPEGylation, and 1:1 to 5:1 for monoPEGylation.

General methods for producing conjugates comprising a polypeptide andwater-soluble polymer moieties are known in the art. See, for example,Karasiewicz et al., U.S. Pat. No. 5,382,657, Greenwald et al., U.S. Pat.No. 5,738, 846, Nieforth et al., Clin. Pharmacol. Ther. 59:636 (1996),Monkarsh et al., Anal. Biochem. 247:434 (1997)). This method can beemployed for making IL-20RA or IL-20RB-comprising homodimeric,heterodimeric or multimeric soluble receptor conjugates.

The present invention contemplates compositions comprising a peptide orpolypeptide described herein. Such compositions can further comprise acarrier. The carrier can be a conventional organic or inorganic carrier.Examples of carriers include water, buffer solution, alcohol, propyleneglycol, macrogol, sesame oil, corn oil, and the like.

7. Isolation of IL-20, IL-20RA and IL-20RB Polypeptides

The polypeptides of the present invention can be purified to at leastabout 80% purity, to at least about 90% purity, to at least about 95%purity, or greater than 95%, such as 96%, 97%, 98%, or greater than 99%purity with respect to contaminating macromolecules, particularly otherproteins and nucleic acids, and free of infectious and pyrogenic agents.The polypeptides of the present invention may also be purified to apharmaceutically pure state, which is greater than 99.9% pure. Incertain preparations, purified polypeptide is substantially free ofother polypeptides, particularly other polypeptides of animal origin.

Fractionation and/or conventional purification methods can be used toobtain preparations of synthetic or recombinant and fusion IL-20,IL-20RA or IL-20RB purified from recombinant host cells, or the samepurified from natural sources (e.g., human tissue sources). In general,ammonium sulfate precipitation and acid or chaotrope extraction may beused for fractionation of samples. Exemplary purification steps mayinclude hydroxyapatite, size exclusion, FPLC and reverse-phase highperformance liquid chromatography. Suitable chromatographic mediainclude derivatized dextrans, agarose, cellulose, polyacrylamide,specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives aresuitable. Exemplary chromatographic media include those mediaderivatized with phenyl, butyl, or octyl groups, such asPhenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate moieties.

Examples of coupling chemistries include cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, hydrazide activation, and carboxyl and amino derivatives forcarbodiimide coupling chemistries. These and other solid media are wellknown and widely used in the art, and are available from commercialsuppliers. Selection of a particular method for polypeptide isolationand purification is a matter of routine design and is determined in partby the properties of the chosen support. See, for example, AffinityChromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988),and Doonan, Protein Purification Protocols (The Humana Press 1996).

Additional variations in isolation and purification of the polypeptidesof the present invention can be devised by those of skill in the art.For example, anti-IL-20, IL-20RA and IL-20RB antibodies, obtained asdescribed below, can be used to isolate large quantities of protein byimmunoaffinity purification.

The polypeptides of the present invention can also be isolated byexploitation of particular properties. For example, immobilized metalion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins, including those comprising polyhistidine tags.Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1 (1985)). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (M.Deutscher, (ed.), Meth. Enzymol. 182:529 (1990)). Within additionalembodiments of the invention, a fusion of the polypeptide of interestand an affinity tag (e.g., maltose-binding protein, an immunoglobulindomain) may be constructed to facilitate purification. Moreover, theligand-binding properties of either the IL-20RA or IL-20RB extracellulardomain can be exploited for purification, for example, ofIL-20RA-comprising soluble receptors; for example, by using affinitychromatography wherein IL-20 ligand is bound to a column and theIL-20RA-comprising receptor is bound and subsequently eluted usingstandard chromatography methods.

The polypeptides of the present invention or fragments thereof may alsobe prepared through chemical synthesis, as described above. Thepolypeptides may be monomers or multimers; glycosylated ornon-glycosylated; PEGylated or non-PEGylated; and may or may not includean initial methionine amino acid residue.

9. Production of Antibodies to IL-20, IL-20RA and IL-20RB Proteins

Antibodies to the polypeptides of the present invention can be obtained,for example, using the product of a IL-20, IL-20RA or IL-20RB expressionvector or any of these polypeptides isolated from a natural source as anantigen. Particularly useful anti-IL-20 antibodies “bind specifically”with IL-20; particularly useful anti-IL-20RA antibodies “bindspecifically” with IL-20RA; while particularly useful anti-IL-20RBantibodies “bind specifically” with IL-20RB. Antibodies are consideredto be specifically binding if the antibodies exhibit at least one of thefollowing two properties: (1) antibodies bind to their specific target(i.e. anti-IL-20 binds to IL-20) with a threshold level of bindingactivity, and (2) antibodies do not significantly cross-react withpolypeptides related to their specific target (i.e. anti-IL-20 does notbind to IL-22).

With regard to the first characteristic, antibodies specifically bind ifthey bind to a polypeptide of the present invention, or peptide orepitope thereof with a binding affinity (K_(a)) of 10⁶ M⁻¹ or greater,preferably 10⁷ M⁻¹ or greater, more preferably 10⁸ M⁻¹ or greater, andmost preferably 109 M-¹ or greater. The binding affinity of an antibodycan be readily determined by one of ordinary skill in the art, forexample, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51:660(1949)). With regard to the second characteristic, antibodies do notsignificantly cross-react with related polypeptide molecules, forexample, if they detect IL-20RA, but not presently known polypeptidesusing a standard Western blot analysis. Examples of known relatedpolypeptides include known cytokine receptors.

Anti-IL-20 antibodies can be produced using antigenic IL-20epitope-bearing peptides and polypeptides. Antigenic epitope-bearingpeptides and polypeptides of the present invention contain a sequence ofat least nine, or between 15 to about 30 amino acids contained withinSEQ ID NO:2 or 3 or another amino acid sequence disclosed herein.However, peptides or polypeptides comprising a larger portion of anamino acid sequence of the invention, containing from 30 to 50 aminoacids, or any length up to and including the entire amino acid sequenceof a polypeptide of the invention, also are useful for inducingantibodies that bind with IL-20. Anti-IL-20RA antibodies can be producedusing antigenic IL-20RA epitope-bearing peptides and polypeptides.Antigenic epitope-bearing peptides and polypeptides of the presentinvention contain a sequence of at least nine, or between 15 to about 30amino acids contained within SEQ ID NO:14 or 15 or another amino acidsequence disclosed herein. However, peptides or polypeptides comprisinga larger portion of an amino acid sequence of the invention, containingfrom 30 to 50 amino acids, or any length up to and including the entireamino acid sequence of a polypeptide of the invention, also are usefulfor inducing antibodies that bind with IL-20. Anti-IL-20RB antibodiescan be produced using antigenic IL-20RB epitope-bearing peptides andpolypeptides. Antigenic epitope-bearing peptides and polypeptides of thepresent invention contain a sequence of at least nine, or between 15 toabout 30 amino acids contained within SEQ ID NO:21 or 23 or anotheramino acid sequence disclosed herein. However, peptides or polypeptidescomprising a larger portion of an amino acid sequence of the invention,containing from 30 to 50 amino acids, or any length up to and includingthe entire amino acid sequence of a polypeptide of the invention, alsoare useful for inducing antibodies that bind with IL-20RB. It isdesirable that the amino acid sequence of the epitope-bearing peptide isselected to provide substantial solubility in aqueous solvents (i.e.,the sequence includes relatively hydrophilic residues, while hydrophobicresidues are typically avoided). Moreover, amino acid sequencescontaining proline residues may be also be desirable for antibodyproduction.

As an illustration, potential antigenic sites in L-20, IL-20RA andIL-20RB were identified using the Jameson-Wolf method, Jameson and Wolf,CABIOS 4:181, (1988), as implemented by the PROTEAN program (version3.14) of LASERGENE (DNASTAR; Madison, Wis.). Default parameters wereused in this analysis.

The Jameson-Wolf method predicts potential antigenic determinants bycombining six major subroutines for protein structural prediction.Briefly, the Hopp-Woods method, Hopp et al., Proc. Nat'l Acad. Sci. USA78:3824 (1981), was first used to identify amino acid sequencesrepresenting areas of greatest local hydrophilicity (parameter: sevenresidues averaged). In the second step, Emini's method, Emini et al., J.Virology 55:836 (1985), was used to calculate surface probabilities(parameter: surface decision threshold (0.6)=1). Third, theKarplus-Schultz method, Karplus and Schultz, Naturwissenschaften 72:212(1985), was used to predict backbone chain flexibility (parameter:flexibility threshold (0.2)=1). In the fourth and fifth steps of theanalysis, secondary structure predictions were applied to the data usingthe methods of Chou-Fasman, Chou, “Prediction of Protein StructuralClasses from Amino Acid Composition,” in Prediction of Protein Structureand the Principles of Protein Conformation, Fasman (ed.), pages 549-586(Plenum Press 1990), and Garnier-Robson, Gamier et al., J. Mol. Biol.120:97 (1978) (Chou-Fasman parameters: conformation table=64 proteins; αregion threshold=103; β region threshold=105; Gamier-Robson parameters:α and β decision constants=0). In the sixth subroutine, flexibilityparameters and hydropathy/solvent accessibility factors were combined todetermine a surface contour value, designated as the “antigenic index.”Finally, a peak broadening function was applied to the antigenic index,which broadens major surface peaks by adding 20, 40, 60, or 80% of therespective peak value to account for additional free energy derived fromthe mobility of surface regions relative to interior regions. Thiscalculation was not applied, however, to any major peak that resides ina helical region, since helical regions tend to be less flexible.

The results of this analysis indicated that the following amino acidsequences of SEQ ID NO:2 would provide suitable antigenic peptides:Hopp/Woods hydrophilicity profiles can be used to determine regions thathave the most antigenic potential within SEQ ID NO:2 (Hopp et al., Proc.Natl. Acad. Sci.78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986and Triquier et al., Protein Engineering 11:153-169, 1998). The profileis based on a sliding six-residue window. Buried G, S, and T residuesand exposed H, Y, and W residues were ignored. Moreover, IL-20 antigenicepitopes within SEQ ID NO:2 as predicted by a Jameson-Wolf plot, e.g.,using DNASTAR Protean program (DNASTAR, Inc., Madison, Wis.) serve aspreferred antigenic epitopes, and can be determined by one of skill inthe art. For Example, neutralizing antibodies to IL-20 includeantibodies, such as neutralizing monoclonal antibodies that can bindIL-20 antigenic epitopes. Accordingly, antigenic epitope-bearingpeptides and polypeptides of IL-20 are useful to raise antibodies thatbind with the IL-20 polypeptides described herein, as well as toidentify and screen anti-IL-20 monoclonal antibodies that areneutralizing, and that may antagonize, reduce, inhibit or block theactivity of IL-20. Such neutralizing monoclonal antibodies of thepresent invention can bind to an IL-20 antigenic epitope. Such epitopeswithin SEQ ID NO:8 as predicted by a Jameson-Wolf plot, e.g., usingDNASTAR Protean program (DNASTAR, Inc., Madison, Wis.) serve aspreferred antigenic epitopes, and can be determined by one of skill inthe art. Such antigenic epitopes include: amino acid residues 42 (Ile)to 102 (Asp) of SEQ ID NO:8; amino acid residues 42 (Ile) to 60 (Ile) ofSEQ ID NO:8; amino acid residues 42 (Ile) to 69 (Glu) of SEQ ID NO:8;amino acid residues 42 (Ile) to 81 (Cys) of SEQ ID NO:8; amino acidresidues 42 (Ile) to 96 (Lys) of SEQ ID NO:8; amino acid residues 42(Ile) to 102 (Asp) of SEQ ID NO:8; amino acid residues 60 (Ile) to 69(Glu) of SEQ ID NO:8; amino acid residues 60 (Ile) to 81 (Cys) of SEQ IDNO:8; amino acid residues 60 (Ile) to 96 (Lys) of SEQ ID NO:8; aminoacid residues 60 (Ile) to 102 (Asp) of SEQ ID NO:8; amino acid residues69 (Glu) to 81 (Cys) of SEQ ID NO:8; amino acid residues 69 (Glu) to 96(Lys) of SEQ ID NO:8; amino acid residues 69 (Glu) to 102 (Asp) of SEQID NO:8; amino acid residues 81 (Cys) to 96 (Lys) of SEQ ID NO:8; aminoacid residues 81 (Cys) to 102 (Asp) of SEQ ID NO:8; and amino acidresidues 96 (Lys) to 102 (Asp) of SEQ ID NO:8.

The results of this analysis indicated that the following amino acidsequences of SEQ ID NO:14 would provide suitable antigenic peptides:amino acid residues 1 (Met) to 9 (Leu) of SEQ ID NO:14; amino acidresidues 1 (Met) to 36 (Gly) of SEQ ID NO:14; amino acid residues 1(Met) to 41 (Ala) of SEQ ID NO:14; amino acid residues 1 (Met) to 58(Pro) of SEQ ID NO:14; amino acid residues 1 (Met) to 63 (Gln) of SEQ IDNO:14; amino acid residues 1 (Met) to 80 (Lys) of SEQ ID NO:14; aminoacid residues 1 (Met) to 94 (Tyr) of SEQ ID NO:14; amino acid residues 1(Met) to 104 (Tyr) of SEQ ID NO:14; amino acid residues 1 (Met) to 120(Cys) of SEQ ID NO:14; amino acid residues 1 (Met) to 128 (Arg) of SEQID NO:14; amino acid residues 1 (Met) to 161 (Trp) of SEQ ID NO:14;amino acid residues 1 (Met) to 169 (Pro) of SEQ ID NO:14; amino acidresidues 1 (Met) to 187 (Asn) of SEQ ID NO:14; amino acid residues 1(Met) to 194 (Trp) of SEQ ID NO:14; amino acid residues 1 (Met) to224(Gly) of SEQ ID NO:14; amino acid residues 1 (Met) to 233 (Glu) ofSEQ ID NO:14; amino acid residues 1 (Met) to 316 (Ile) of SEQ ID NO:14;amino acid residues 1 (Met) to 323 (Ile) of SEQ ID NO:14; amino acidresidues 1 (Met) to 335 (Asp) of SEQ ID NO:14; amino acid residues 1(Met) to 340 (Asn) of SEQ ID NO:14; amino acid residues 1 (Met) to 354(Glu) of SEQ ID NO:14; amino acid residues 1 (Met) to 371 (Cys) of SEQID NO:14; amino acid residues 1 (Met) to381 (Ser) of SEQ ID NO:14; aminoacid residues 1 (Met) to 384 (Gln) of SEQ ID NO:14; amino acid residues1 (Met) to 397 (Thr) of SEQ ID NO:14; amino acid residues 1 (Met) to 412(Ala) of SEQ ID NO:14; amino acid residues 1 (Met) to 418 (Glu) of SEQID NO:14; amino acid residues 1 (Met) to 462 (Gln) of SEQ ID NO:14;amino acid residues 1 (Met) to 476 (Ser) of SEQ ID NO:14; amino acidresidues 1 (Met) to 483 (Asp) of SEQ ID NO:14; amino acid residues 1(Met) to 486 (Thr) of SEQ ID NO:14; amino acid residues 1 (Met) to 496(Ser) of SEQ ID NO:14; amino acid residues 1 (Met) to 511 (Gly) of SEQID NO:14; amino acid residues 1 (Met) to 523 (Glu) of SEQ ID NO:14;amino acid residues 1 (Met) to 536 (Thr) of SEQ ID NO:14; amino acidresidues 36 (Gly) to 63(Gln) of SEQ ID NO:14; amino acid residues 36(Gly) to 94 (tyr) of SEQ ID NO:14; amino acid residues 36 (Gly) to 128(Arg) of SEQ ID NO:14; amino acid residues 36 (Gly) to 169 (Pro) of SEQID NO:14; amino acid residues 36 (Gly) to 194 (Trp) of SEQ ID NO:14;amino acid residues 36 (Gly) to 233 (Glu) of SEQ ID NO:14; amino acidresidues 36 (Gly) to 323 (Ser) of SEQ ID NO:14; amino acid residues 36(Gly) to 340 (Asn) of SEQ ID NO:14; amino acid residues 36 (Gly) to 354(Glu) of SEQ ID NO:14; amino acid residues 36 (Gly) to 381 (Ser) of SEQID NO:14; amino acid residues 36 (Gly) to 397 (Thr) of SEQ ID NO:14;amino acid residues 36 (Gly) to 418 (Glu) of SEQ ID NO:14; amino acidresidues 36 (Gly) to 476 (Ser) of SEQ ID NO:14; amino acid residues 36(Gly) to 486 (Thr) of SEQ ID NO:14; amino acid residues 36 (Gly) to 511(Gly) of SEQ ID NO:14; amino acid residues 36 (Gly) to 536 (Thr) of SEQID NO:14; amino acid residues 58 (Pro) to 63 (Gln) of SEQ ID NO:14;amino acid residues 58 (Pro) to 94 (tyr) of SEQ ID NO:14; amino acidresidues 58 (Pro) to 128 (Arg) of SEQ ID NO:14; amino acid residues 58(Pro) to 169 (Pro) of SEQ ID NO:14; amino acid residues 58 (Pro) to 194(Trp) of SEQ ID NO:14; amino acid residues 58 (Pro) to 233 (Glu) of SEQID NO:14; amino acid residues 58 (Pro) to 323 (Ser) of SEQ ID NO:14;amino acid residues 58 (Pro) to 340 (Asn) of SEQ ID NO:14; amino acidresidues 58 (Pro) to 354 (Glu) of SEQ ID NO:14; amino acid residues 58(Pro) to 381 (Ser) of SEQ ID NO:14; amino acid residues 58 (Pro) to 397(Thr) of SEQ ID NO:14; amino acid residues 58 (Pro) to 418 (Glu) of SEQID NO:14; amino acid residues 58 (Pro) to 476 (Ser) of SEQ ID NO:14;amino acid residues 58 (Pro) to 486 (Thr) of SEQ ID NO:14; amino acidresidues 58 (Pro) to 511 (Gly) of SEQ ID NO:14; amino acid residues 58(Pro) to 536 (Thr) of SEQ ID NO:14; amino acid residues 80 (Lys) to 94(tyr) of SEQ ID NO:14; amino acid residues 80 (Lys) to 128 (Arg) of SEQID NO:14; amino acid residues 80 (Lys) to 169 (Pro) of SEQ ID NO:14;amino acid residues 80 (Lys) to 194 (Trp) of SEQ ID NO:14; amino acidresidues 80 (Lys) to 233 (Glu) of SEQ ID NO:14; amino acid residues 80(Lys) to 323 (Ser) of SEQ ID NO:14; amino acid residues 80 (Lys) to 340(Asn) of SEQ ID NO:14; amino acid residues 80 (Lys) to 354 (Glu) of SEQID NO:14; amino acid residues 80 (Lys) to 381 (Ser) of SEQ ID NO:14;amino acid residues 80 (Lys) to 397 (Thr) of SEQ ID NO:14; amino acidresidues 80 (Lys) to 418 (Glu) of SEQ ID NO:14; amino acid residues 80(Lys) to 476 (Ser) of SEQ ID NO:14; amino acid residues 80 (Lys) to 486(Thr) of SEQ ID NO:14; amino acid residues 80 (Lys) to 511 (Gly) of SEQID NO:14; amino acid residues 80 (Lys) to 536 (Thr) of SEQ ID NO:14;amino acid residues 120 (Cys) to 128 (Arg) of SEQ ID NO:14; amino acidresidues 120 (Cys) to 169 (Pro) of SEQ ID NO:14; amino acid residues 120(Cys) to 194 (Trp) of SEQ ID NO:14; amino acid residues 120 (Cys) to 233(Glu) of SEQ ID NO:14; amino acid residues 120 (Cys) to 323 (Ser) of SEQID NO:14; amino acid residues 120 (Cys) to 340 (Asn) of SEQ ID NO:14;amino acid residues 120 (Cys) to 354 (Glu) of SEQ ID NO:14; amino acidresidues 120 (Cys) to 381 (Ser) of SEQ ID NO:14; amino acid residues 120(Cys) to 397 (Thr) of SEQ ID NO:14; amino acid residues 120 (Cys) to 418(Glu) of SEQ ID NO:14; amino acid residues 120 (Cys) to 476 (Ser) of SEQID NO:14; amino acid residues 120 (Cys) to 486 (Thr) of SEQ ID NO:14;amino acid residues 120 (Cys) to 511 (Gly) of SEQ ID NO:14; amino acidresidues 120 (Cys) to 536 (Thr) of SEQ ID NO:14; amino acid residues 161(Trp) to 169 (Pro) of SEQ ID NO:14; amino acid residues 161 (Trp) to 194(Trp) of SEQ ID NO:14; amino acid residues 161 (Trp) to 233 (Glu) of SEQID NO:14; amino acid residues 161 (Trp) to 323 (Ser) of SEQ ID NO:14;amino acid residues 161 (Trp) to 340 (Asn) of SEQ ID NO:14; amino acidresidues 161 (Trp) to 354 (Glu) of SEQ ID NO:14; amino acid residues 161(Trp) to 381 (Ser) of SEQ ID NO:14; amino acid residues 161 (Trp) to 397(Thr) of SEQ ID NO:14; amino acid residues 161 (Trp) to 418 (Glu) of SEQID NO:14; amino acid residues 161 (Trp) to 476 (Ser) of SEQ ID NO:14;amino acid residues 161 (Trp) to 486 (Thr) of SEQ ID NO:14; amino acidresidues 161 (Trp) to 511 (Gly) of SEQ ID NO:14; amino acid residues 161(Trp) to 536 (Thr) of SEQ ID NO:14; amino acid residues 187 (Asn) to 194(Trp) of SEQ ID NO:14; amino acid residues 187 (Asn) to 233 (Glu) of SEQID NO:14; amino acid residues 187 (Asn) to 323 (Ser) of SEQ ID NO:14;amino acid residues 187 (Asn) to 340 (Asn) of SEQ ID NO:14; amino acidresidues 187 (Asn) to 354 (Glu) of SEQ ID NO:14; amino acid residues 187(Asn) to 381 (Ser) of SEQ ID NO:14; amino acid residues 187 (Asn) to 397(Thr) of SEQ ID NO:14; amino acid residues 187 (Asn) to 418 (Glu) of SEQID NO:14; amino acid residues 187 (Asn) to 476 (Ser) of SEQ ID NO:14;amino acid residues 187 (Asn) to 486 (Thr) of SEQ ID NO:14; amino acidresidues 187 (Asn) to 511 (Gly) of SEQ ID NO:14; amino acid residues 187(Asn) to 536 (Thr) of SEQ ID NO:14; amino acid residues 224 (Gly) to 233(Glu) of SEQ ID NO:14; amino acid residues 224 (Gly) to 323 (Ser) of SEQID NO:14; amino acid residues 224 (Gly) to 340 (Asn) of SEQ ID NO:14;amino acid residues 224 (Gly) to 354 (Glu) of SEQ ID NO:14; amino acidresidues 224 (Gly) to 381 (Ser) of SEQ ID NO:14; amino acid residues 224(Gly) to 397 (Thr) of SEQ ID NO:14; amino acid residues 224 (Gly) to 418(Glu) of SEQ ID NO:14; amino acid residues 224 (Gly) to 476 (Ser) of SEQID NO:14; amino acid residues 224 (Gly) to 486 (Thr) of SEQ ID NO:14;amino acid residues 224 (Gly) to 511 (Gly) of SEQ ID NO:14; amino acidresidues 224 (Gly) to 536 (Thr) of SEQ ID NO:14; amino acid residues 316(Ile) to 323 (Ser) of SEQ ID NO:14; amino acid residues 316 (Ile) to 340(Asn) of SEQ ID NO:14; amino acid residues 316 (Ile) to 354 (Glu) of SEQID NO:14; amino acid residues 316 (Ile) to 381 (Ser) of SEQ ID NO:14;amino acid residues 316 (Ile) to 397 (Thr) of SEQ ID NO:14; amino acidresidues 316 (Ile) to 418 (Glu) of SEQ I) NO:14; amino acid residues 316(Ile) to 476 (Ser) of SEQ ID NO:14; amino acid residues 316 (Ile) to 486(Thr) of SEQ ID NO:14; amino acid residues 316 (Ile) to 511 (Gly) of SEQID NO:14; amino acid residues 316 (Ile) to 536 (Thr) of SEQ ID NO:14;amino acid residues 335 (Asp) to 340 (Asn) of SEQ ID NO:14; amino acidresidues 335 (Asp) to 354 (Glu) of SEQ ID NO:14; amino acid residues 335(Asp) to 381 (Ser) of SEQ ID NO:14; amino acid residues 335 (Asp) to 397(Thr) of SEQ ID NO:14; amino acid residues 335 (Asp) to 418 (Glu) of SEQID NO:14; amino acid residues 335 (Asp) to 476 (Ser) of SEQ ID NO:14;amino acid residues 335 (Asp) to 486 (Thr) of SEQ ID NO:14; amino acidresidues 335 (Asp) to 511 (Gly) of SEQ ID NO:14; amino acid residues 335(Asp) to 536 (Thr) of SEQ ID NO:14; amino acid residues 371 (Cys) to 381(Ser) of SEQ ID NO:14; amino acid residues 371(Cys) to 397 (Thr) of SEQID NO:14; amino acid residues 371(Cys) to 418 (Glu) of SEQ ID NO:14;amino acid residues 371 (Cys) to 476 (Ser) of SEQ ID NO:14; amino acidresidues 371 (Cys) to 486 (Thr) of SEQ ID NO:14; amino acid residues371(Cys) to 511 (Gly) of SEQ ID NO:14; amino acid residues 371(Cys) to536 (Thr) of SEQ ID NO:14; amino acid residues 384 (Gln) to 397 (Thr) ofSEQ ID NO:14; amino acid residues 384 (Gin) to 418 (Glu) of SEQ IDNO:14; amino acid residues 384 (Gln) to 476 (Ser) of SEQ ID NO:14; aminoacid residues 384 (Gln) to 486 (Thr) of SEQ ID NO:14; amino acidresidues 384 (Gln) to 511 (Gly) of SEQ ID NO:14; amino acid residues 384(Gln) to 536 (Thr) of SEQ ID NO:14; amino acid residues 412 (Ala) to 418(Glu) of SEQ ID NO:14; amino acid residues 412 (Ala) to 476 (Ser) of SEQID NO:14; amino acid residues 412 (Ala) to 486 (Thr) of SEQ ID NO:14;amino acid residues 412 (Ala) to 511 (Gly) of SEQ ID NO:14; amino acidresidues 412 (Ala) to 536 (Thr) of SEQ ID NO:14; amino acid residues 462(Gln) to 476 (Ser) of SEQ ID NO:14; amino acid residues 462 (Gln) to 486(Thr) of SEQ ID NO:14; amino acid residues 462 (Gln) to 511 (Gly) of SEQID NO:14; amino acid residues 462 (Gln) to 536 (Thr) of SEQ ID NO:14;amino acid residues 483 (Asp) to 486 (Thr) of SEQ ID NO:14; amino acidresidues 483 (Asp) to 511 (Gly) of SEQ ID NO:14; amino acid residues 483(Asp) to 536 (Thr) of SEQ ID NO:14; amino acid residues 496 (Ser) to 511(Gly) of SEQ ID NO:14; amino acid residues 496 (Ser) to 536 (Thr) of SEQID NO:14; amino acid residues 523 (Glu) to 536 (Thr) of SEQ ID NO:14.

The results of this analysis indicated that the following amino acidsequences of SEQ ID NO:14 would provide suitable antigenic peptides:L-20RB antigenic epitopes within SEQ ID NO:21 include: amino acidresidues 70 (Tyr) to 74 (Tyr) of SEQ ID NO:21; amino acid residues 70(Tyr) to 101 (Asp) of SEQ ID NO:21; amino acid residues 70 (Tyr) to 135(Ser) of SEQ ID NO:21; amino acid residues 70 (Tyr) to 178 (Glu) of SEQID NO:21; amino acid residues 70 (Tyr) to 283 (Lys) of SEQ ID NO:21;amino acid residues 92 (Thr) to 101 (Asp) of SEQ ID NO:21; amino acidresidues 92 (Thr) to 135 (Ser) of SEQ ID NO:21; amino acid residues 92(Thr) to 178 (Glu) of SEQ ID NO:21; amino acid residues 92 (Thr) to 283(Lys) of SEQ ID NO:21; amino acid residues 130 (Pro) to 135 (Ser) of SEQID NO:21; amino acid residues 130 (Pro) to 178 (Glu) of SEQ ID NO:21;amino acid residues 130 (Pro) to 283 (Lys) of SEQ ID NO:21; amino acidresidues 171 (Arg) to 178 (Glu) of SEQ ID NO:21; amino acid residues 171(Arg) to 283 (Lys) of SEQ ID NO:21; amino acid residues 279 (Asn) to 283(Lys) of SEQ ID NO:21.Moreover, suitable antigens also include theIL-20RA or IL-20RB polypeptides comprising a IL-20RA or IL-20RB cytokinebinding, or extracellular domain disclosed above in combination withanother class I or II cytokine extracellular domain, such as those thatform soluble IL-20RA and/or IL-20RB heterodimeric or multimericpolypeptides.

Polyclonal antibodies to recombinant polypeptides of the presentinvention or to those same polypeptides isolated from natural sourcescan be prepared using methods well-known to those of skill in the art.See, for example, Green et al., “Production of Polyclonal Antisera,” inImmunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992),and Williams et al., “Expression of foreign proteins in E. coli usingplasmid vectors and purification of specific polyclonal antibodies,” inDNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),page 15 (Oxford University Press 1995). The immunogenicity of apolypeptide of the present invention can be increased through the use ofan adjuvant, such as alum (aluminum hydroxide) or Freund's complete orincomplete adjuvant. Polypeptides useful for immunization also includefusion polypeptides, such as fusions of IL-20RA or IL-20RB or a portionthereof with an immunoglobulin polypeptide or with maltose bindingprotein. The polypeptide immunogen may be a full-length molecule or aportion thereof. If the polypeptide portion is “hapten-like,” suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

Although polyclonal antibodies are typically raised in animals such ashorses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs, goats, orsheep, an antibody of the present invention may also be derived from asubhuman primate antibody. General techniques for raising diagnosticallyand therapeutically useful antibodies in baboons may be found, forexample, in Goldenberg et al., international patent publication No. WO91/11465, and in Losman et al., Int. J. Cancer 46:310 (1990).

Alternatively, monoclonal anti-IL-20, IL-20RA or IL-20RB antibodies canbe generated. Rodent monoclonal antibodies to specific antigens may beobtained by methods known to those skilled in the art (see, for example,Kohler et al., Nature 256:495 (1975), Coligan et al. (eds.), CurrentProtocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991) [“Coligan”], Picksley et al., “Production of monoclonal antibodiesagainst proteins expressed in E. coli,” in DNA Cloning 2: ExpressionSystems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford UniversityPress 1995)).

Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising a IL-20, IL-20RA or IL-20RB gene product,verifying the presence of antibody production by removing a serumsample, removing the spleen to obtain B-lymphocytes, fusing theB-lymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones which produce antibodies to theantigen, culturing the clones that produce antibodies to the antigen,and isolating the antibodies from the hybridoma cultures.

In addition, an anti-IL-20, anti-IL-20RA or anti-IL-20RB antibody of thepresent invention may be derived from a human monoclonal antibody. Humanmonoclonal antibodies are obtained from transgenic mice that have beenengineered to produce specific human antibodies in response to antigenicchallenge. In this technique, elements of the human heavy and lightchain locus are introduced into strains of mice derived from embryonicstem cell lines that contain targeted disruptions of the endogenousheavy chain and light chain loci. The transgenic mice can synthesizehuman antibodies specific for human antigens, and the mice can be usedto produce human antibody-secreting hybridomas. Methods for obtaininghuman antibodies from transgenic mice are described, for example, byGreen et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994).

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography (see, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).

For particular uses, it may be desirable to prepare fragments of any ofthe anti-IL-20, anti-IL-20RA or anti-IL-20RB antibodies. Such antibodyfragments can be obtained, for example, by proteolytic hydrolysis of theantibody. Antibody fragments can be obtained by pepsin or papaindigestion of whole antibodies by conventional methods. As anillustration, antibody fragments can be produced by enzymatic cleavageof antibodies with pepsin to provide a 5S fragment denoted F(ab′)₂. Thisfragment can be further cleaved using a thiol reducing agent to produce3.5S Fab′ monovalent fragments. Optionally, the cleavage reaction can beperformed using a blocking group for the sulfhydryl groups that resultfrom cleavage of disulfide linkages. As an alternative, an enzymaticcleavage using pepsin produces two monovalent Fab fragments and an Fcfragment directly. These methods are described, for example, byGoldenberg, U.S. Pat. No. 4,331,647, Nisonoff et al., Arch Biochem.Biophys. 89:230 (1960), Porter, Biochem. J. 73:119 (1959), Edelman etal., in Methods in Enzymology Vol. 1, page 422 (Academic Press 1967),and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association can be noncovalent, as described by Inbar etal., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde (see, for example,Sandhu, Crit. Rev. Biotech. 12:437 (1992)).

The Fv fragments may comprise V_(H) and V_(L) chains which are connectedby a peptide linker. These single-chain antigen binding proteins (scFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains which are connected by anoligonucleotide. The structural gene is inserted into an expressionvector which is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are described, for example, by Whitlow et al., Methods: ACompanion to Methods in Enzymology 2:97 (1991) (also see, Bird et al.,Science 242:423 (1988), Ladner et al., U.S. Pat. No. 4,946,778, Pack etal., Bio/Technology 11:1271 (1993), and Sandhu, supra).

As an illustration, a scFV can be obtained by exposing lymphocytes to apolypeptide of the present invention in vitro, and selecting antibodydisplay libraries in phage or similar vectors (for instance, through useof immobilized or labeled Il-20, IL-20RA or IL-20RB protein or peptide).Genes encoding polypeptides having potential IL-20, IL-20RA or IL-20RBpolypeptide binding domains can be obtained by screening random peptidelibraries displayed on phage (phage display) or on bacteria, such as E.coli. Nucleotide sequences encoding the polypeptides can be obtained ina number of ways, such as through random mutagenesis and randompolynucleotide synthesis. These random peptide display libraries can beused to screen for peptides which interact with a known target which canbe a protein or polypeptide, such as a ligand or receptor, a biologicalor synthetic macromolecule, or organic or inorganic substances.Techniques for creating and screening such random peptide displaylibraries are known in the art (Ladner et al., U.S. Pat. No. 5,223,409,Ladner et al., U.S. Pat. No. 4,946,778, Ladner et al., U.S. Pat. No.5,403,484, Ladner et al., U.S. Pat. No. 5,571,698, and Kay et al., PhageDisplay of Peptides and Proteins (Academic Press, Inc. 1996)) and randompeptide display libraries and kits for screening such libraries areavailable commercially, for instance from CLONTECH Laboratories, Inc.(Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New EnglandBiolabs, Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc.(Piscataway, N.J.). Random peptide display libraries can be screenedusing the IL-20, IL-20RA and IL-20RB sequences disclosed herein toidentify proteins which bind to IL-20.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106 (1991),Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies: Production, Engineering and Clinical Application,Ritter et al. (eds.), page 166 (Cambridge University Press 1995), andWard et al., “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137 (Wiley-Liss, Inc. 1995)).

Alternatively, an anti-IL-20, anti-IL-20RA or anti-IL-20RB antibody maybe derived from a “humanized” monoclonal antibody. Humanized monoclonalantibodies are produced by transferring mouse complementary determiningregions from heavy and light variable chains of the mouse immunoglobulininto a human variable domain. Typical residues of human antibodies arethen substituted in the framework regions of the murine counterparts.The use of antibody components derived from humanized monoclonalantibodies obviates potential problems associated with theimmunogenicity of murine constant regions. General techniques forcloning murine immunoglobulin variable domains are described, forexample, by Orlandi et al., Proc. Nat'l Acad. Sci. USA 86:3833 (1989).Techniques for producing humanized monoclonal antibodies are described,for example, by Jones et al., Nature 321:522 (1986), Carter et al.,Proc. Nat'l Acad. Sci. USA 89:4285 (1992), Sandhu, Crit. Rev. Biotech.12:437 (1992), Singer et al., J. Immun. 150:2844 (1993), Sudhir (ed.),Antibody Engineering Protocols (Humana Press, Inc. 1995), Kelley,“Engineering Therapeutic Antibodies,” in Protein Engineering: Principlesand Practice, Cleland et al. (eds.), pages 399-434 (John Wiley & Sons,Inc. 1996), and by Queen et al., U.S. Pat. No. 5,693,762 (1997).

Polyclonal anti-idiotype antibodies can be prepared by immunizinganimals with anti-IL-20, anti-IL-20RA or anti-IL-20RB antibodies orantibody fragments, using standard techniques. See, for example, Greenet al., “Production of Polyclonal Antisera,” in Methods In MolecularBiology: Immunochemical Protocols, Manson (ed.), pages 1-12 (HumanaPress 1992). Also, see Coligan at pages 2.4.1-2.4.7. Alternatively,monoclonal anti-idiotype antibodies can be prepared using anti-IL-20,anti-IL-20RA or anti-]]L-20RB antibodies or antibody fragments asimmunogens with the techniques, described above. As another alternative,humanized anti-idiotype antibodies or subhuman primate anti-idiotypeantibodies can be prepared using the above-described techniques. Methodsfor producing anti-idiotype antibodies are described, for example, byIrie, U.S. Pat. No. 5,208,146, Greene, et. al., U.S. Pat. No. 5,637,677,and Varthakavi and Minocha, J. Gen. Virol. 77:1875 (1996).

An anti-IL-20, anti-IL-20RA or anti-IL-20RB antibody can be conjugatedwith a detectable label to form an anti-IL-20, anti-IL-20RA oranti-IL-20RB immunoconjugate. Suitable detectable labels include, forexample, a radioisotope, a fluorescent label, a chemiluminescent label,an enzyme label, a bioluminescent label or colloidal gold. Methods ofmaking and detecting such detectably-labeled immunoconjugates arewell-known to those of ordinary skill in the art, and are described inmore detail below.

The detectable label can be a radioisotope that is detected byautoradiography. Isotopes that are particularly useful for the purposeof the present invention are ³H, ¹²⁵I, ¹³¹I, ³⁵S and ¹⁴C.

Anti-IL-20, anti-IL-20RA or anti-IL-20RB immunoconjugates can also belabeled with a fluorescent compound. The presence of afluorescently-labeled antibody is determined by exposing theimmunoconjugate to light of the proper wavelength and detecting theresultant fluorescence. Fluorescent labeling compounds includefluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin,allophycocyanin, o-phthal-dehyde and fluorescamine.

Alternatively, anti-IL-20, anti-IL-20RA or anti-IL-20RB immunoconjugatescan be detectably labeled by coupling an antibody component to achemiluminescent compound. The presence of the chemiluminescent-taggedimmunoconjugate is determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofchemiluminescent labeling compounds include luminol, isoluminol, anaromatic acridinium ester, an imidazole, an acridinium salt and anoxalate ester.

Similarly, a bioluminescent compound can be used to label anti-IL-20,anti-IL-20RA or anti-IL-20RB immunoconjugates of the present invention.Bioluminescence is a type of chemiluminescence found in biologicalsystems in which a catalytic protein increases the efficiency of thechemiluminescent reaction. The presence of a bioluminescent protein isdetermined by detecting the presence of luminescence. Bioluminescentcompounds that are useful for labeling include luciferin, luciferase andaequorin.

Alternatively, anti-IL-20, anti-IL-20RA or anti-IL-20RB immunoconjugatescan be detectably labeled by linking an anti-IL-20, anti-IL-20RA oranti-IL-20RB antibody component to an enzyme. When the antibody-enzymeconjugate is incubated in the presence of the appropriate substrate, theenzyme moiety reacts with the substrate to produce a chemical moietywhich can be detected, for example, by spectrophotometric, fluorometricor visual means. Examples of enzymes that can be used to detectablylabel polyspecific immunoconjugates include β-galactosidase, glucoseoxidase, peroxidase and alkaline phosphatase.

Those of skill in the art will know of other suitable labels which canbe employed in accordance with the present invention. The binding ofmarker moieties to anti-IL-20, anti-IL-20RA or anti-IL-20RB antibodiescan be accomplished using standard techniques known to the art. Typicalmethodology in this regard is described by Kennedy et al., Clin. Chim.Acta 70:1 (1976), Schurs et al., Clin. Chim. Acta 81:1 (1977), Shih etal., Int'l J. Cancer 46:1101 (1990), Stein et al., Cancer Res. 50:1330(1990), and Coligan, supra.

Moreover, the convenience and versatility of immunochemical detectioncan be enhanced by using anti-IL-20, anti-IL-20RA or anti-IL-20RBantibodies that have been conjugated with avidin, streptavidin, andbiotin (see, for example, Wilchek et al. (eds.), “Avidin-BiotinTechnology,” Methods In Enzymology, Vol. 184 (Academic Press 1990), andBayer et al., “Immunochemical Applications of Avidin-Biotin Technology,”in Methods In Molecular Biology, Vol. 10, Manson (ed.), pages 149-162(The Humana Press, Inc. 1992).

Methods for performing immunoassays are well-established. See, forexample, Cook and Self, “Monoclonal Antibodies in DiagnosticImmunoassays,” in Monoclonal Antibodies: Production, Engineering, andClinical Application, Ritter and Ladyman (eds.), pages 180-208,(Cambridge University Press, 1995), Perry, “The Role of MonoclonalAntibodies in the Advancement of Immunoassay Technology,” in MonoclonalAntibodies: Principles and Applications, Birch and Lennox (eds.), pages107-120 (Wiley-Liss, Inc. 1995), and Diamandis, Immunoassay (AcademicPress, Inc. 1996).

The present invention also contemplates kits for performing animmunological diagnostic assay for IL-20, IL-20RA or IL-20RB geneexpression. Such kits comprise at least one container comprising ananti-IL-20, anti-IL-20RA or anti-IL-20RB antibody, or antibody fragment.A kit may also comprise a second container comprising one or morereagents capable of indicating the presence of IL-20, IL-20RA or IL-20RBantibody or antibody fragments. Examples of such indicator reagentsinclude detectable labels such as a radioactive label, a fluorescentlabel, a chemiluminescent label, an enzyme label, a bioluminescentlabel, colloidal gold, and the like. A kit may also comprise a means forconveying to the user that IL-20, IL-20RA or IL-20RB antibodies orantibody fragments are used to detect the corresponding protein. Forexample, written instructions may state that the enclosed antibody orantibody fragment can be used to detect IL-20, IL-20RA or IL-20RB. Thewritten material can be applied directly to a container, or the writtenmaterial can be provided in the form of a packaging insert.

8. Use of Anti-IL-20, Anti-IL-20RA or Anti-IL-20RB Antibodies toAntagonize IL-20RA and IL-20RB Binding to IL-20

Alternative techniques for generating or selecting antibodies usefulherein include in vitro exposure of lymphocytes to soluble IL-20RA orIL-20RB receptor polypeptides or fragments thereof, such as antigenicepitopes, and selection of antibody display libraries in phage orsimilar vectors (for instance, through use of immobilized or labeledsoluble IL-20RA or IL-20RB receptor polypeptides or fragments thereof,such as antigenic epitopes). Genes encoding polypeptides havingpotential binding domains such as soluble IL-20RA or IL-20RB receptorpolypeptides or fragments thereof, such as antigenic epitopes can beobtained by screening random peptide libraries displayed on phage (phagedisplay) or on bacteria, such as E. coli. Nucleotide sequences encodingthe polypeptides can be obtained in a number of ways, such as throughrandom mutagenesis and random polynucleotide synthesis. These randompeptide display libraries can be used to screen for peptides thatinteract with a known target that can be a protein or polypeptide, suchas a ligand or receptor, a biological or synthetic macromolecule, ororganic or inorganic substances. Techniques for creating and screeningsuch random peptide display libraries are known in the art (Ladner etal., U.S. Pat. No. 5,223,409; Ladner et al., U.S. Pat. No. 4,946,778;Ladner et al., U.S. Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No.5,571,698) and random peptide display libraries and kits for screeningsuch libraries are available commercially, for instance from Clontech(Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New EnglandBiolabs, Inc. (Beverly, Mass.) and Pharmacia LKB Biotechnology Inc.(Piscataway, N.J.). Random peptide display libraries can be screenedusing the soluble IL-20RA and/or IL-20RB receptor polypeptides orfragments thereof, such as antigenic epitope polypeptide sequencesdisclosed herein to identify proteins which bind to IL-20RA orIL-20RB-comprising receptor polypeptides. These “binding polypeptides,”which interact with soluble IL-20RA and/or IL-20RB-comprising receptorpolypeptides, can be used for tagging cells; for isolating homologpolypeptides by affinity purification; they can be directly orindirectly conjugated to drugs, toxins, radionuclides and the like.These binding polypeptides can also be used in analytical methods suchas for screening expression libraries and neutralizing activity, e.g.,for blocking interaction between IL-20 ligand and receptor, or viralbinding to a receptor. The binding polypeptides can also be used fordiagnostic assays for determining circulating levels of soluble IL-20RAand/or IL-20RB-comprising receptor polypeptides; for detecting orquantitating soluble or non-soluble IL-20RA-comprising receptors asmarker of underlying pathology or disease. These binding polypeptidescan also act as “antagonists” to block soluble or membrane-bound IL-20RAor IL-20RB monomeric receptor or IL-20RA/IL-20RB homodimeric,heterodimeric or multimeric polypeptide binding (e.g. to ligand) andsignal transduction in vitro and in vivo. Again, these bindingpolypeptides serve as anti-IL-20RA or anti-IL-20RB monomeric receptor oranti-IL-20RA and/or anti-IL-20RB homodimeric, heterodimeric ormultimeric polypeptides and are useful for inhibiting IL-20 activity, aswell as receptor activity or protein-binding. Antibodies raised to thenatural receptor complexes of the present invention, and IL-20, IL-20RAand IL-20RB-epitope-binding antibodies, and anti-IL-20, IL-20RA orIL-20RB neutralizing monoclonal antibodies may be preferred embodiments,as they may act more specifically and can inhibit IL-20. Moreover, theantagonistic and binding activity of the antibodies of the presentinvention can be assayed in an IL-20 proliferation, signal trap,luciferase or binding assays in the presence of 1IL-20, and IL-20RAand/or IL-20RB-comprising soluble receptors, and other biological orbiochemical assays described herein.

Antibodies to soluble IL-20RA, IL-20RB and IL-20RB/IL-20RB receptorpolypeptides or fragments thereof, such as antigenic epitopes may beused for inhibiting the inflammatory effects of IL-20 in vivo, fortheraputic use against psoriasis, endotoxemia, arthritis, asthma, IBD,colitis, psoriatic arthritis, rheumatoid arthritis or otherIL-20-induced inflammatory conditions; tagging cells that expressIL-20RA, IL-20RB and IL-20RB/IL-20RB receptors; for isolating solubleIL-2OR and/or IL-20RB-comprising receptor polypeptides by affinitypurification; for diagnostic assays for determining circulating levelsof soluble IL-20RA and/or IL-20RB-comprising receptor polypeptides; fordetecting or quantitating soluble IL-20RA and/or IL-20RB-comprisingreceptors as marker of underlying pathology or disease; in analyticalmethods employing FACS; for screening expression libraries; forgenerating anti-idiotypic antibodies that can act as IL-20 agonists; andas neutralizing antibodies or as antagonists to block IL-20RA and/orIL-20RB receptor function, or to block IL-20 activity in vitro and invivo. Suitable direct tags or labels include radionuclides, enzymes,substrates, cofactors, biotin, inhibitors, fluorescent markers,chemiluminescent markers, magnetic particles and the like; indirect tagsor labels may feature use of biotin-avidin or othercomplement/anti-complement pairs as intermediates. Antibodies herein mayalso be directly or indirectly conjugated to drugs, toxins,radionuclides and the like, and these conjugates used for in vivodiagnostic or therapeutic applications. Moreover, antibodies to solubleIL-20RA and/or IL-20RB-comprising receptor polypeptides, or fragmentsthereof may be used in vitro to detect denatured or non-denaturedIL-20RA and/or IL-20RB-comprising receptor polypeptides or fragmentsthereof in assays, for example, Western Blots or other assays known inthe art.

Antibodies to soluble IL-20RA and/or IL-20RB receptor or soluble IL-20RAor IL-20RB homodimeric, heterodimeric or multimeric receptorpolypeptides are useful for tagging cells that express the correspondingreceptors and assaying their expression levels, for affinitypurification, within diagnostic assays for determining circulatinglevels of receptor polypeptides, analytical methods employingfluorescence-activated cell sorting. Moreover, divalent antibodies, andanti-idiotypic antibodies may be used as agonists to mimic the effect ofIL-20.

Antibodies herein can also be directly or indirectly conjugated todrugs, toxins, radionuclides and the like, and these conjugates used forin vivo diagnostic or therapeutic applications. For instance, antibodiesor binding polypeptides which recognize soluble IL-20RA and/or IL-20RBreceptor or soluble IL-20RA or IL-20RB homodimeric, heterodimeric ormultimeric receptor polypeptides can be used to identify or treattissues or organs that express a corresponding anti-complementarymolecule (for example, a IL-20RA-comprising soluble or membrane-boundreceptor). More specifically, antibodies to soluble IL-20RA and/orIL-20RB-comprising receptor polypeptides, or bioactive fragments orportions thereof, can be coupled to detectable or cytotoxic moleculesand delivered to a mammal having cells, tissues or organs that expressthe IL-20RA, IL-20RB or IL-20RA/IL-20RB-comprising receptor such asIL-20RA and/or IL-20RB -expressing cancers.

Suitable detectable molecules may be directly or indirectly attached topolypeptides that bind IL-20RA and/or IL-20RB-comprising receptorpolypeptides, such as “binding polypeptides,” (including bindingpeptides disclosed above), antibodies, or bioactive fragments orportions thereof. Suitable detectable molecules include radionuclides,enzymes, substrates, cofactors, inhibitors, fluorescent markers,chemiluminescent markers, magnetic particles and the like. Suitablecytotoxic molecules may be directly or indirectly attached to thepolypeptide or antibody, and include bacterial or plant toxins (forinstance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and thelike), as well as therapeutic radionuclides, such as iodine-131,rhenium-188 or yttrium-90 (either directly attached to the polypeptideor antibody, or indirectly attached through means of a chelating moiety,for instance). Binding polypeptides or antibodies may also be conjugatedto cytotoxic drugs, such as adriamycin. For indirect attachment of adetectable or cytotoxic molecule, the detectable or cytotoxic moleculecan be conjugated with a member of a complementary/anticomplementarypair, where the other member is bound to the binding polypeptide orantibody portion. For these purposes, biotin/streptavidin is anexemplary complementary/anticomplementary pair.

In another embodiment, binding polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat cancer cells or tissues).Alternatively, if the binding polypeptide has multiple functionaldomains (i.e., an activation domain or a ligand binding domain, plus atargeting domain), a fusion protein including only the targeting domainmay be suitable for directing a detectable molecule, a cytotoxicmolecule or a complementary molecule to a cell or tissue type ofinterest. In instances where the fusion protein including only a singledomain includes a complementary molecule, the anti-complementarymolecule can be conjugated to a detectable or cytotoxic molecule. Suchdomain-complementary molecule fusion proteins thus represent a generictargeting vehicle for cell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates.

In another embodiment, IL-20, IL-20RA or IL-20RB bindingpolypeptide-cytokine or antibody-cytokine fusion proteins can be usedfor enhancing in vivo killing of target tissues (for example, spleen,pancreatic, blood, lymphoid, colon, and bone marrow cancers), if thebinding polypeptide-cytokine or anti-IL-20, IL-20RA or IL-20RB receptorantibody targets the hyperproliferative cell (See, generally, Hornick etal., Blood 89:4437-47, 1997). The described fusion proteins enabletargeting of a cytokine to a desired site of action, thereby providingan elevated local concentration of cytokine. Suitable anti-IL-20,anti-IL-20RA or anti-IL-20RB monomer, homodimer, heterodimer or multimerantibodies target an undesirable cell or tissue (i.e., a tumor or aleukemia), and the fused cytokine mediates improved target cell lysis byeffector cells. Suitable cytokines for this purpose include interleukin2 and granulocyte-macrophage colony-stimulating factor (GM-CSF), forinstance.

Alternatively, IL-20, IL-20RA or IL-20RB receptor binding polypeptidesor antibody fusion proteins described herein can be used for enhancingin vivo killing of target tissues by directly stimulating a IL-20,IL-20RA or IL-20RB receptor-modulated apoptotic pathway, resulting incell death of hyperproliferative cells expressing IL-20 receptors.

9. Therapeutic Uses of Polypeptides Having IL-20, IL-20RA or IL-20RBActivity or Antibodies Thereto

Amino acid sequences having soluble IL-20RA and/or IL-20RB activity canbe used to modulate the immune system by binding IL-20, and thus,preventing the binding of IL-20 with endogenous IL-20RA/IL-20RB. 11-20antagonists, such as anti-IL-20, anti-IL-20RA and anti-IL-20RBantibodies, can also be used to modulate the immune system by inhibitingthe binding of IL-20 with the endogenous receptor. Accordingly, thepresent invention includes the use of proteins, polypeptides, andpeptides having anti-IL-20 activity (such as soluble IL-20RA, IL-20RB orIL-20RA/IL-20RB polypeptides, polypeptide fragments, analogs and fusionproteins) to a subject which lacks an adequate amount of thispolypeptide, or which produces an excess of IL-20. IL-20RA and IL-20RBantagonists (e.g., anti-IL-20RA and IL-20RB antibodies) can be also usedto treat a subject which produces an excess of either IL-20 or IL-20RAand/or IL-20RB. Suitable subjects include mammals, such as humans. Suchpolypeptides and antibodies are useful in antagonizing IL-20, in thetreatment of psoriasis, psoriatic arthritis, arthritis, endotoxemia,inflammatory bowel disease (IBD), colitis, and other inflammatoryconditions disclosed herein.

Two lines of evidence indicate that a role IL-20 and its receptor areinvolved in psoriasis. This multigenic skin disease is characterized byincreased keratinocyte proliferation, altered keratinocytedifferentiation, and infiltration of immune cells into the skin. Thefirst line of evidence for a role of IL-20 in psoriasis is that theobserved hyperkeratosis and thickened epidermis in the transgenic micethat resemble human psoriatic abnormalities. Decreased numbers oftonofilaments, thought to be related to defective keratinization, are astriking feature of human psoriasis. Intramitochondrial inclusions havebeen found in both chemically induced and naturally occurringhyperplastic skin conditions in mice. The cause of the inclusions andtheir effects on mitochondrial function, if any, are unknown. Weconclude that IL-20 transgenic mice exhibit many of the characteristicsobserved in human psoriasis.

A second line of evidence that implicates the IL-20 receptor inpsoriasis is that both IL-20RA and IL-20RB mRNA are markedly upregulatedin human psoriatic skin compared to normal skin. Both IL-20 receptorsubunits are expressed in keratinocytes throughout the epidermis and arealso expressed in a subset of immune and endothelial cells. We proposethat increased expression of an activated IL-20 receptor may alter theinteractions between endothelial cells, immune cells and keratinocytes,leading to dysregulation of keratinocyte proliferation anddifferentiation.

Moreover, IL-20 stimulates signal transduction in the human keratinocyteHaCaT cell line, supporting a direct action of this novel ligand inskin. In addition, IL-1β, EGF and TNF-α, proteins known to be active inkeratinocytes and to be involved with proliferative and pro-inflammatorysignals in skin, enhance the response to IL-20. In both HaCaT and BHKcells expressing the IL-20 receptor, IL-20 signals through STAT3.

As indicated in the discussion above and the examples below, IL-20 isinvolved in the pathology of psoriasis. The present invention is inparticular a method for treating psoriasis by administering antagoniststo IL-20. The antagonists to IL-20 can either be a soluble receptor thatbinds to IL-20, such a soluble IL-20RA, IL-20RB or IL-20RA/IL-20RB, orantibodies, single chain antibodies or fragments of antibodies that bindto either IL-20 or either a subunit such as IL-20RA or IL-20RB or theIL-20 receptor as a whole. The antagonists will thus prevent activationof the IL-20 receptor.

Psoriasis is one of the most common dermatologic diseases, affecting upto 1 to 2 percent of the world's population. It is a chronicinflammatory skin disorder characterized by erythematous, sharplydemarcated papules and rounded plaques, covered by silvery micaceousscale. The skin lesions of psoriasis are variably pruritic. Traumatizedareas often develop lesions of psoriasis. Additionally, other externalfactors may exacerbate psoriasis including infections, stress, andmedications, e.g. lithium, beta blockers, and anti-malarials.

The most common variety of psoriasis is called plaque type. Patientswith plaque-type psoriasis will have stable, slowly growing plaques,which remain basically unchanged for long periods of time. The mostcommon areas for plaque psoriasis to occur are the elbows knees, glutealcleft, and the scalp. Involvement tends to be symmetrical. Inversepsoriasis affects the intertriginous regions including the axilla,groin, submammary region, and navel, and it also tends to affect thescalp, palms, and soles. The individual lesions are sharply demarcatedplaques but may be moist due to their location. Plaque-type psoriasisgenerally develops slowly and runs an indolent course. It rarelyspontaneously remits.

Eruptive psoriasis (guttate psoriasis) is most common in children andyoung adults. It develops acutely in individuals without psoriasis or inthose with chronic plaque psoriasis. Patients present with many smallerythematous, scaling papules, frequently after upper respiratory tractinfection with beta-hemolytic streptococci. Patients with psoriasis mayalso develop pustular lesions. These may be localized to the palms andsoles or may be generalized and associated with fever, malaise,diarrhea, and arthralgias.

About half of all patients with psoriasis have fingernail involvement,appearing as punctate pitting, nail thickening or subungualhyperkeratosis. About 5 to 10 percent of patients with psoriasis haveassociated joint complaints, and these are most often found in patientswith fingernail involvement. Although some have the coincidentoccurrence of classic rheumatoid arthritis, many have joint disease thatfalls into one of five type associated with psoriasis: (1) diseaselimited to a single or a few small joints (70 percent of cases); (2) aseronegative rheumatoid arthritis-like disease; (3) involvement of thedistal interphalangeal joints; (4) severe destructive arthritis with thedevelopment of “arthritis mutilans”; and (5) disease limited to thespine.

Psoriasis can be treated by administering antagonists to IL-20. Thepreferred antagonists are either a soluble receptor to IL-20 orantibodies, antibody fragments or single chain antibodies that bind toIL-20, IL-20RA or IL-20RB. Such antagonists can be administered alone orin combination with other established therapies such as lubricants,keratolytics, topical corticosteroids, topical vitamin D derivatives,anthralin, systemic antimetabolites such as methotrexate,psoralen-ultraviolet-light therapy (PUVA), etretinate, isotretinoin,cyclosporine, and the topical vitamin D3 derivative calcipotriol.Moreover, such antagonists can be administered to individualsubcutaneously, intravenously, or transdermally using a cream ortransdermal patch that contains the antagonist. If administeredsubcutaneously, the antagonist can be injected into one or morepsoriatic plaques. If administered transdermally, the antagonists can beadministered directly on the plaques using a cream, ointment, salve, orsolution containing the antagonist.

Antagonists to IL-20 can be administered to a person who has asthma,bronchitis or cystic fibrosis or other inflammatory lung disease totreat the disease. The antagonists can be administered by any suitablemethod including intravenous, subcutaneous, bronchial lavage, and theuse of inhalant containing the antagonist. Particular embodiments of thepresent invention are directed toward use of soluble IL-20RA and/orL-20RB and anti-IL-20, anti-IL-20RA or anti-IL-20RB antibodies asantagonists in inflammatory and immune diseases or conditions such aspsoriasis, psoriatic arthritis, rheumatoid arthritis, inflammatory boweldisease (IBD), Crohn's Disease, diverticulosis, asthma, pancreatitis,type I diabetes (IDDM), pancreatic cancer, pancreatitis, Graves Disease,colon and intestinal cancer, autoimmune disease, sepsis, organ or bonemarrow transplant; inflammation due to endotoxemia, trauma, surgery orinfection; amyloidosis; splenomegaly; graft versus host disease; andwhere inhibition of inflammation, immune suppression, reduction ofproliferation of hematopoietic, immune, inflammatory or lymphoid cells,macrophages, T-cells (including Th1 and Th2 cells), suppression ofimmune response to a pathogen or antigen, or other instances whereinhibition of IL-20 cytokines is desired.

Moreover, antibodies or binding polypeptides that bind IL-20, IL-20RA orIL-20RB polypeptides described herein, are useful to:

1) Antagonize or block signaling via IL-20 receptors in the treatment ofacute inflammation, inflammation as a result of trauma, tissue injury,surgery, sepsis or infection, and chronic inflammatory diseases such asasthma, inflammatory bowel disease (IBD), chronic colitis, splenomegaly,rheumatoid arthritis, recurrent acute inflammatory episodes (e.g.,tuberculosis), and treatment of amyloidosis, and atherosclerosis,Castleman's Disease, asthma, and other diseases associated with theinduction of acute-phase response.

2) Antagonize or block signaling via IL-20 receptors in the treatment ofautoimmune diseases such as IDDM, multiple sclerosis (MS), systemicLupus erythematosus (SLE), myasthenia gravis, rheumatoid arthritis, andIBD to prevent or inhibit signaling in immune cells (e.g. lymphocytes,monocytes, leukocytes). Alternatively antibodies, such as monoclonalantibodies (MAb) to an y of IL-20, IL-20RA, or IL-20RB-comprisingreceptors, can also be used as an antagonist to deplete unwanted immunecells to treat autoimmune disease. Asthma, allergy and other atopicdisease may be treated with an MAb against, for example, IL-20, toinhibit the immune response or to deplete offending cells. Blocking orinhibiting signaling via IL-20, IL-20RA or IL-20RB, using thepolypeptides and antibodies of the present invention, may also benefitdiseases of the pancreas, kidney, pituitary and neuronal cells. IDDM,NIDDM, pancreatitis, and pancreatic carcinoma may benefit. Polypeptidesof the present invention may serve as a target for MAb therapy of cancerwhere an antagonizing MAb inhibits cancer growth and targetsimmune-mediated killing. (Holliger P, and Hoogenboom, H: Nature Biotech.16: 1015-1016, 1998). Mabs to soluble IL-20RA may also be useful totreat nephropathies such as glomerulosclerosis, membranous neuropathy,amyloidosis (which also affects the kidney among other tissues), renalarteriosclerosis, glomerulonephritis of various origins,fibroproliferative diseases of the kidney, as well as kidney dysfunctionassociated with SLE, IDDM, type II diabetes (NIDDM), renal tumors andother diseases.

3) Agonize or initiate signaling via IL-20 receptors in the treatment ofautoimmune diseases such as IDDM, MS, SLE, myasthenia gravis, rheumatoidarthritis, and IBD. Anti-IL-20, anti-IL-20RA or anti-IL-20RBneutralizing and monoclonal antibodies may signal lymphocytes or otherimmune cells to differentiate, alter proliferation, or change productionof cytokines or cell surface proteins that ameliorate autoimmunity.Specifically, modulation of a T-helper cell response to an alternatepattern of cytokine secretion may deviate an autoimmune response toameliorate disease (Smith J A et al., J. Immunol. 160:4841-4849, 1998).

The soluble polypeptides described herein can be used toneutralize/block IL-20 or IL-20 activity, either singly or together, inthe treatment of autoimmune disease, atopic disease, NIDDM, pancreatitisand kidney dysfunction as described above. A soluble form of IL-20RA,IL-20RB and/or IL-20RA/IL-20RB may be used to promote an antibodyresponse mediated by Th cells and/or to promote the production of IL-4or other cytokines by lymphocytes or other immune cells.

The soluble IL-20RA, IL-20RB and/or IL-20RA/IL-20RB -comprisingreceptors of the present invention are useful as antagonists of IL-20cytokine. Such antagonistic effects can be achieved by directneutralization or binding of IL-20. In addition to antagonistic uses,the soluble receptors of the present invention can bind IL-20 and act ascarrier proteins for IL-20, in order to transport the Ligand todifferent tissues, organs, and cells within the body. As such, thesoluble receptors of the present invention can be fused or coupled tomolecules, polypeptides or chemical moieties that direct thesoluble-receptor-Ligand complex to a specific site, such as a tissue,specific immune cell, or tumor. For example, in acute infection or somecancers, benefit may result from induction of inflammation and localacute phase response proteins by the action of IL-20. Thus, the solublereceptors of the present invention can be used to specifically directthe action of IL-20. See, Cosman, D. Cytokine 5: 95-106, 1993; andFernandez-Botran, R. Exp. Opin. Invest. Drugs 9:497-513, 2000.

Moreover, the soluble receptors of the present invention can be used tostabilize IL-20, to increase the bioavailability, therapeutic longevity,and/or efficacy of the Ligand by stabilizing the Ligand from degradationor clearance, or by targeting the ligand to a site of action within thebody. For example the naturally occurring IL-6/soluble IL-6R complexstabilizes IL-6 and can signal through the gp130 receptor. See, Cosman,D. supra., and Fernandez-Botran, R. supra. Furthermore the complexes mayhave distinct pharmacokinetic properties such as affecting half-life,dose/response and organ or tissue specificity. IL-20RA/IL-20 orIL-20RB/IL-20 complexes thus may have agonist activity to enhance animmune response or stimulate mesangial cells or to stimulate hepaticcells. Alternatively only tissues expressing a signaling subunit theheterodimerizes with the complex may be affected analogous to theresponse to IL6/IL6R complexes (Hirota H. et al., Proc. Nat'l. Acad.Sci. 92:4862-4866, 1995; Hirano, T. in Thomason, A. (Ed.) “The CytokineHandbook”, 3^(rd) Ed., p. 208-209). Soluble receptor/cytokine complexesfor IL12 and CNTF display similar activities.

Moreover Inflammation is a protective response by an organism to fendoff an invading agent. Inflammation is a cascading event that involvesmany cellular and humoral mediators. On one hand, suppression ofinflammatory responses can leave a host immunocompromised; however, ifleft unchecked, inflammation can lead to serious complications includingchronic inflammatory diseases (e.g., psoriasis, arthritis, rheumatoidarthritis, multiple sclerosis, inflammatory bowel disease and the like),septic shock and multiple organ failure. Importantly, these diversedisease states share common inflammatory mediators. The collectivediseases that are characterized by inflammation have a large impact onhuman morbidity and mortality. Therefore it is clear thatanti-inflammatory proteins, such as IL-20, IL-20RA and IL-20RB, andanti-IL-20, anti-IL-20RA and anti-IL-20RB antibodies, could have crucialtherapeutic potential for a vast number of human and animal diseases,from asthma and allergy to autoimmunity and septic shock.

1. Arthritis

Arthritis, including osteoarthritis, rheumatoid arthritis, arthriticjoints as a result of injury, and the like, are common inflammatoryconditions which would benefit from the therapeutic use ofanti-inflammatory proteins, such as those polypeptides of the presentinvention. For Example, rheumatoid arthritis (RA) is a systemic diseasethat affects the entire body and is one of the most common forms ofarthritis. It is characterized by the inflammation of the membranelining the joint, which causes pain, stiffness, warmth, redness andswelling. Inflammatory cells release enzymes that may digest bone andcartilage. As a result of rheumatoid arthritis, the inflamed jointlining, the synovium, can invade and damage bone and cartilage leadingto joint deterioration and severe pain amongst other physiologiceffects. The involved joint can lose its shape and alignment, resultingin pain and loss of movement.

Rheumatoid arthritis (RA) is an immune-mediated disease particularlycharacterized by inflammation and subsequent tissue damage leading tosevere disability and increased mortality. A variety of cytokines areproduced locally in the rheumatoid joints. Numerous studies havedemonstrated that IL-1 and TNF-alpha, two prototypic pro-inflammatorycytokines, play an important role in the mechanisms involved in synovialinflammation and in progressive joint destruction. Indeed, theadministration of TNF-alpha and IL-1 inhibitors in patients with RA hasled to a dramatic improvement of clinical and biological signs ofinflammation and a reduction of radiological signs of bone erosion andcartilage destruction. However, despite these encouraging results, asignificant percentage of patients do not respond to these agents,suggesting that other mediators are also involved in the pathophysiologyof arthritis (Gabay, Expert. Opin. Biol. Ther. 2(2):135-149, 2002). Oneof those mediators could be IL-20, and as such a molecule that binds orinhibits IL-20 activity, such as IL-20RA or IL-20RB polypeptides, oranti-IL-20 antibodies or binding partners, could serve as a valuabletherapeutic to reduce inflammation in rheumatoid arthritis, and otherarthritic diseases.

There are several animal models for rheumatoid arthritis known in theart. For example, in the collagen-induced arthritis (CIA) model, micedevelop chronic inflammatory arthritis that closely resembles humanrheumatoid arthritis. Since CIA shares similar immunological andpathological features with RA, this makes it an ideal model forscreening potential human anti-inflammatory compounds. The CIA model isa well-known model in mice that depends on both an immune response, andan inflammatory response, in order to occur. The immune responsecomprises the interaction of B-cells and CD4+ T-cells in response tocollagen, which is given as antigen, and leads to the production ofanti-collagen antibodies. The inflammatory phase is the result of tissueresponses from mediators of inflammation, as a consequence of some ofthese antibodies cross-reacting to the mouse's native collagen andactivating the complement cascade. An advantage in using the CIA modelis that the basic mechanisms of pathogenesis are known. The relevantT-cell and B-cell epitopes on type II collagen have been identified, andvarious immunological (e.g., delayed-type hypersensitivity andanti-collagen antibody) and inflammatory (e.g., cytokines, chemokines,and matrix-degrading enzymes) parameters relating to immune-mediatedarthritis have been determined, and can thus be used to assess testcompound efficacy in the CIA model (Wooley, Curr. Opin. Rheum. 3:407-20,1999; Williams et al., Immunol. 89:9784-788, 1992; Myers et al., LifeSci. 61:1861-78, 1997; and Wang et al., Immunol. 92:8955-959, 1995).

2. Endotoxemia

Endotoxemia is a severe condition commonly resulting from infectiousagents such as bacteria and other infectious disease agents, sepsis,toxic shock syndrome, or in immunocompromised patients subjected toopportunistic infections, and the like. Therapeutically useful ofanti-inflammatory proteins, such as polypeptides and antibodies of thepresent invention, could aid in preventing and treating endotoxemia inhumans and animals. IL-20, IL-20RA or IL-20RB polypeptides, anti-IL-20,anti-IL-20RA or anti-IL-20RB antibodies, could serve as a valuabletherapeutic to reduce inflammation and pathological effects inendotoxemia.

Lipopolysaccharide (LPS) induced endotoxemia engages many of theproinflammatory mediators that produce pathological effects in theinfectious diseases and LPS induced endotoxemia in rodents is a widelyused and acceptable model for studying the pharmacological effects ofpotential pro-inflammatory or immunomodulating agents. LPS, produced ingram-negative bacteria, is a major causative agent in the pathogenesisof septic shock (Glausner et al., Lancet 338:732, 1991). A shock-likestate can indeed be induced experimentally by a single injection of LPSinto animals. Molecules produced by cells responding to LPS can targetpathogens directly or indirectly. Although these biological responsesprotect the host against invading pathogens, they may also cause harm.Thus, massive stimulation of innate immunity, occurring as a result ofsevere Gram-negative bacterial infection, leads to excess production ofcytokines and other molecules, and the development of a fatal syndrome,septic shock syndrome, which is characterized by fever, hypotension,disseminated intravascular coagulation, and multiple organ failure(Dumitru et al. Cell 103:1071-1083, 2000).

These toxic effects of LPS are mostly related to macrophage activationleading to the release of multiple inflammatory mediators. Among thesemediators, TNF appears to play a crucial role, as indicated by theprevention of LPS toxicity by the administration of neutralizinganti-TNF antibodies (Beutler et al., Science 229:869, 1985). It is wellestablished that lug injection of E. coli LPS into a C57B1/6 mouse willresult in significant increases in circulating IL-6, TNF-alpha, IL-1,and acute phase proteins (for example, SAA) approximately 2 hours postinjection. The toxicity of LPS appears to be mediated by these cytokinesas passive immunization against these mediators can result in decreasedmortality (Beutler et al., Science 229:869, 1985). The potentialimmunointervention strategies for the prevention and/or treatment ofseptic shock include anti-TNF mAb, IL-1 receptor antagonist, LIF, IL-10,and G-CSF.

3. Inflammatory Bowel Disease (IBD)

In the United States approximately 500,000 people suffer fromInflammatory Bowel Disease (IBD) which can affect either colon andrectum (Ulcerative colitis) or both, small and large intestine (Crohn'sDisease). The pathogenesis of these diseases is unclear, but theyinvolve chronic inflammation of the affected tissues. IL-20, IL-20RA orIL-20RB polypeptides, anti-IL-20, anti-IL-20RA or anti-IL-20RBantibodies or binding partners, could serve as a valuable therapeutic toreduce inflammation and pathological effects in IBD and relateddiseases.

Ulcerative colitis (UC) is an inflammatory disease of the largeintestine, commonly called the colon, characterized by inflammation andulceration of the mucosa or innermost lining of the colon. Thisinflammation causes the colon to empty frequently, resulting indiarrhea. Symptoms include loosening of the stool and associatedabdominal cramping, fever and weight loss. Although the exact cause ofUC is unknown, recent research suggests that the body's natural defensesare operating against proteins in the body which the body thinks areforeign (an “autoimmune reaction”). Perhaps because they resemblebacterial proteins in the gut, these proteins may either instigate orstimulate the inflammatory process that begins to destroy the lining ofthe colon. As the lining of the colon is destroyed, ulcers formreleasing mucus, pus and blood. The disease usually begins in the rectalarea and may eventually extend through the entire large bowel. Repeatedepisodes of inflammation lead to thickening of the wall of the intestineand rectum with scar tissue. Death of colon tissue or sepsis may occurwith severe disease. The symptoms of ulcerative colitis vary in severityand their onset may be gradual or sudden. Attacks may be provoked bymany factors, including respiratory infections or stress.

Although there is currently no cure for UC available, treatments arefocused on suppressing the abnormal inflammatory process in the colonlining. Treatments including corticosteroids immunosuppressives (e.g.azathioprine, mercaptopurine, and methotrexate) and aminosalicytates areavailable to treat the disease. However, the long-term use ofimmunosuppressives such as corticosteroids and azathioprine can resultin serious side effects including thinning of bones, cataracts,infection, and liver and bone marrow effects. In the patients in whomcurrent therapies are not successful, surgery is an option. The surgeryinvolves the removal of the entire colon and the rectum.

There are several animal models that can partially mimic chroniculcerative colitis. The most widely used model is the2,4,6-trinitrobenesulfonic acid/ethanol (TNBS) induced colitis model,which induces chronic inflammation and ulceration in the colon. WhenTNBS is introduced into the colon of susceptible mice via intra-rectalinstillation, it induces T-cell mediated immune response in the colonicmucosa, in this case leading to a massive mucosal inflammationcharacterized by the dense infiltration of T-cells and macrophagesthroughout the entire wall of the large bowel. Moreover, thishistopathologic picture is accompanies by the clinical picture ofprogressive weight loss (wasting), bloody diarrhea, rectal prolapse, andlarge bowel wall thickening (Neurath et al. Intern. Rev. Immunol.19:51-62, 2000).

Another colitis model uses dextran sulfate sodium (DSS), which inducesan acute colitis manifested by bloody diarrhea, weight loss, shorteningof the colon and mucosal ulceration with neutrophil infiltration.DSS-induced colitis is characterized histologically by infiltration ofinflammatory cells into the lamina propria, with lymphoid hyperplasia,focal crypt damage, and epithelial ulceration. These changes are thoughtto develop due to a toxic effect of DSS on the epithelium and byphagocytosis of lamina propria cells and production of TNF-alpha andIFN-gamma. Despite its common use, several issues regarding themechanisms of DSS about the relevance to the human disease remainunresolved. DSS is regarded as a T cell-independent model because it isobserved in T cell-deficient animals such as SCID mice.

4. Psoriasis

Psoriasis is a chronic skin condition that affects more than sevenmillion Americans. Psoriasis occurs when new skin cells grow abnormally,resulting in inflamed, swollen, and scaly patches of skin where the oldskin has not shed quickly enough. Plaque psoriasis, the most commonform, is characterized by inflamed patches of skin (“lesions”) toppedwith silvery white scales. Psoriasis may be limited to a few plaques orinvolve moderate to extensive areas of skin, appearing most commonly onthe scalp, knees, elbows and trunk. Although it is highly visible,psoriasis is not a contagious disease. The pathogenesis of the diseasesinvolves chronic inflammation of the affected tissues. IL-20, IL-20RA orIL-20RB polypeptides, anti-IL-20, anti-IL-20RA or anti-IL-20RBantibodies or binding partners, could serve as a valuable therapeutic toreduce inflammation and pathological effects in psoriasis, otherinflammatory skin diseases, skin and mucosal allergies, and relateddiseases.

Psoriasis is a T-cell mediated inflammatory disorder of the skin thatcan cause considerable discomfort. It is a disease for which there is nocure and affects people of all ages. Psoriasis affects approximately twopercent of the populations of European and North America. Althoughindividuals with mild psoriasis can often control their disease withtopical agents, more than one million patients worldwide requireultraviolet or systemic immunosuppressive therapy. Unfortunately, theinconvenience and risks of ultraviolet radiation and the toxicities ofmany therapies limit their long-term use. Moreover, patients usuallyhave recurrence of psoriasis, and in some cases rebound, shortly afterstopping immunosuppressive therapy.

IL-20 is a novel IL-10 homologue that was shown to cause neonatallethality with skin abnormalities including aberrant epidermaldifferentiation in IL-20 transgenic mice (Blumberg H et al., Cell104:9-19, 2001) IL-20 receptor is dramatically upregulated in psoriaticskin. Moreover, over expression of IL-20 was shown in human psoriaticlesions, suggesting that IL-20 is involved in human psoriasis. Moreover,as described herein, over expression of L-20 in transgenic mice showedepidermal thickening and immune cell involvement indicative of apsoriatic phenotype. As such, antagonists to IL-20 activity, such asIL-20RA, IL-20RB and/or IL-20RA/IL-20RB soluble receptors and antibodiesthereto including the anti-human-IL-20, anti-human-IL-20RA andanti-human-IL-20RB monoclonal and neutralizing antibodies of the presentinvention, are useful in therapeutic treatment of inflammatory diseases,particularly as antagonists to IL-20 in the treatment of psoriasis.Moreover, antagonists to IL-20 activity, such as IL-20RA, IL-20RB and/orIL-20RA/IL-20RB soluble receptors and antibodies thereto including theanti-human-IL-20, anti-human-IL-20RA and anti-human-IL-20RB monoclonaland neutralizing antibodies of the present invention, are useful intherapeutic treatment of other inflammatory diseases for example asantagonists to IL-20 in the treatment of atopic dermatitis, IBD,colitis, Endotoxemia, arthritis, rheumatoid arthritis, and psoriaticarthritis adult respiratory disease (ARD), septic shock, multiple organfailure, inflammatory lung injury such as asthma or bronchitis,bacterial pneumonia, psoriasis, eczema, atopic and contact dermatitis,and inflammatory bowel disease such as ulcerative colitis and Crohn'sdisease.

Moreover, the soluble receptors and antibodies of the present inventioncan be used in the prevention and therapy against weight loss associatedwith a number of inflammatory diseases described herein, as well as forcancer (e.g., chemotherapy and cachexia), and infectious diseases. Forexample, severe weight loss is a key marker associated with models forsepticemia, MS, RA, and tumor models. In addition, weight loss is a keyparameter for many human diseases including cancer, infectious diseaseand inflammatory disease. Weight loss was shown in mice injected withIL-22Adenovirus described herein. Anti-IL-20 antibodies and IL-20antagonists such as the soluble receptors and antibodies of the presentinvention, can be tested for their ability to prevent and treat weightloss in mice injected with IL-20 adenoviruses described herein. Methodsof determining a prophylactic or therapeutic regimen for such IL-20antagonists is known in the art and can be determined using the methodsdescribed herein.

The soluble receptors and antibodies of the present invention may alsobe used within diagnostic systems for the detection of circulatinglevels of IL-20, and in the detection of IL-20 associated with acutephase inflammatory response. Within a related embodiment, antibodies orother agents that specifically bind to the polypeptides and solublereceptors of the present invention can be used to detect circulatingreceptor polypeptides; conversely, IL-20RA, IL-20RB or IL-20RA/IL-20RBsoluble receptors themselves can be used to detect circulating orlocally-acting IL-20 polypeptides. Elevated or depressed levels ofligand or receptor polypeptides may be indicative of pathologicalconditions, including inflammation or cancer. IL-20 is known to induceassociated acute phase inflammatory response. Moreover, detection ofacute phase proteins or molecules such as IL-20 can be indicative of achronic inflammatory condition in certain disease states (e.g.,psoriasis, rheumatoid arthritis, colitis, IBD). Detection of suchconditions serves to aid in disease diagnosis as well as help aphysician in choosing proper therapy.

In utero administration of neutralizing anti-IL-20 antibodies can beused to show efficacy in vivo in disease models by reducing oreliminating the skin phenotype found in IL-20 transgenic pups which overexpress IL-20. There are precedents in the art for in utero treatmentwith neutralizing monoclonal antibodies (mAbs). In one case, thedevelopment of the B-1 subset of B cells was dramatically affected bytreating pregnant female mice with a mAb specific for the Bcell-specific molecule, CD19 (e.g., Krop I. Et al., Eur. J. Immunol.26(1):238-42, 1996). Krop et al. injected timed pregnant miceintraperitoneally with 500 ug of rat anti-mouse CD19 mAb (or a ratisotype-matched control Ab) in PBS beginning on day 9 of gestation, withsubsequent injections every other day until birth. Pups were alsoinjected once with 500 ug of these antibodies at 10 days of age. Inanother case, Tanaka et al., found that in utero treatment withmonoclonal antibody to IL-2 receptor beta-chain completely abrogatesdevelopment of Thy-1+ dendritic epidermal cells. The two distinctsubunits of the IL-2 receptor, i.e. the alpha-chain (IL-2R alpha) andthe beta-chain (IL-2R beta), are expressed in an almost mutuallyexclusive fashion throughout fetal thymus ontogeny. Blocking IL-2R beta,a signal transducing component of IL-2R, by administering a neutralizingmAb to IL-2R beta, resulted in the complete and selective disappearanceof Thy-1+ skin dendritic epidermal cells. Development of any other Tcell subsets was uncompromised. This indicated that IL-2 plays a crucialrole in the development of fetal V gamma 5+ cells and their descendants(see, Tanaka, T. et al., Int Immunol. 4(4):487-9, 1992). In addition,Schattemann G C et al., showed that PDGF-A is required for normal murinecardiovascular development using an in utero system. Several lines ofevidence suggest that platelet-derived growth factor A chain (PDGF-A) isrequired for normal embryonic cardiovascular development. Introductionof anti-PDGF-A neutralizing antibodies into mouse deciduas in uteroresulted in the selective disruption of PDGF-A ligand-receptorinteractions in vivo for a period of 18-24 hr and allowed assessment ofwhether PDGF-A is required for cardiovascular development and when it isrequired (see, Schattemann G C et al., Dev. Biol. 176(1):133-42, 1996).These results, as well as others described in the art, provide evidencethat neutralizing mAbs can elicit strong effects in utero. Similarly,data showing the efficacy of neutralizing IL-20 with monoclonalantibodies in vivo in disease models to reduce or eliminate the skinphenotype found in IL-20 transgenic pups which over express IL-20respectively can be shown. These transgenic mice are born with a “shiny”skin appearance, due at least in part to a thickening of the epidermisas described herein. The IL-20 TG pups expressing fairly low levels ofthe transgenic cytokine can recover and do survive to breed.

In addition to other disease models described herein, the activity ofanti-IL-20, anti-IL-20RA and anti-IL-20RB antibodies on inflammatorytissue derived from human psoriatic lesions can be measured in vivousing a severe combined immune deficient (SCID) mouse model. Severalmouse models have been developed in which human cells are implanted intoimmunodeficient mice (collectively referred to as xenograft models);see, for example, Cattan A R, Douglas E, Leuk. Res. 18:513-22, 1994 andFlavell, D J, Hematological Oncology 14:67-82, 1996. As an in vivoxenograft model for psoriasis, human psoriatic skin tissue is implantedinto the SCID mouse model, and challenged with an appropriateantagonist. Anti-IL-20, anti-IL-20RA and anti-IL-20RB antibodies thatblock the activity of IL-20 are preferred antagonists, however, solubleIL-20RA, as well as other IL-20 blocking antagonists can be used in thismodel. Similarly, tissues or cells derived from human colitis, IBD,arthritis, or other inflammatory lesions can be used in the SCID modelto assess the anti-inflammatory properties of the IL-20 antagonistsdescribed herein.

Therapies designed to abolish, retard, or reduce inflammation usinganti-IL-20, anti-IL-20RA and anti-IL-20RB antibodies antibodies or itsderivatives, agonists, conjugates or variants can be tested byadministration of these antibodies or soluble IL-20RA, IL-20RB orIL-20RA/IL-20RB compounds to SCID mice bearing human inflammatory tissue(e.g., psoriatic lesions and the like). Efficacy of treatment ismeasured and statistically evaluated as increased anti-inflammatoryeffect within the treated population over time using methods well knownin the art. Some exemplary methods include, but are not limited tomeasuring for example, in a psoriasis model, epidermal thickness, thenumber of inflammatory cells in the upper dermis, and the grades ofparakeratosis. Such methods are known in the art and described herein.For example, see Zeigler, M. et al. Lab Invest 81:1253, 2001; Zollner,T. M. et al. J. Clin. Invest. 109:671, 2002; Yamanaka, N. et al.Microbio.l Immunol. 45:507, 2001; Raychaudhuri, S. P. et al. Br. J.Dermatol. 144:931, 2001; Boehncke, W. H et al. Arch. Dermatol. Res.291:104, 1999; Boehncke, W. H et al. J. Invest. Dermatol. 116:596, 2001;Nickoloff, B. J. et al. Am. J. Pathol. 146:580, 1995; Boehncke, W. H etal. J. Cutan. Pathol. 24:1, 1997; Sugai, J., M. et al. J. Dermatol. Sci.17:85, 1998; and Villadsen L. S. et al. J. Clin. Invest. 112:1571, 2003.Inflammation may also be monitored over time using well-known methodssuch as flow cytometry (or PCR) to quantitate the number of inflammatoryor lesional cells present in a sample, score (weight loss, diarrhea,rectal bleeding, colon length) for IBD, paw disease score andinflammation score for CIA RA model. For example, therapeutic strategiesappropriate for testing in such a model include direct treatment usinganti-IL-20, anti-IL-20RA and anti-IL-20RB antibodies, other IL-20antagonists, or related conjugates or antagonists based on thedisrupting interaction of anti-IL-20, anti-IL-20RA or anti-IL-20RBantibodies with IL-20, or for cell-based therapies utilizing anti-IL-20,anti-IL-20RA and anti-IL-20RB antibodies or its derivatives, agonists,conjugates or variants.

Moreover, psoriasis is a chronic inflammatory skin disease that isassociated with hyperplastic epidermal keratinocytes and infiltratingmononuclear cells, including CD4+ memory T cells, neutrophils andmacrophages (Christophers, Int. Arch. Allergy Immunol., 110:199, 1996).It is currently believed that environmental antigens play a significantrole in initiating and contributing to the pathology of the disease.However, it is the loss of tolerance to self-antigens that is thought tomediate the pathology of psoriasis. Dendritic cells and CD4⁺ T cells arethought to play an important role in antigen presentation andrecognition that mediate the immune response leading to the pathology.We have recently developed a model of psoriasis based on the CD4+CD45RBtransfer model (Davenport et al., Internat. Immunopharmacol.,2:653-672). Anti-IL-20, anti-IL-20RA or anti-IL-20RB, or solubleIL-20RA, IL-20RB or IL-20RA/IL-20RB, are administered to the mice.Inhibition of disease scores (skin lesions, inflammatory cytokines)indicates the effectiveness of IL-20 antagonists in psoriasis, e.g.,anti-IL-20, anti-IL-20RA or anti-IL-20RB antibodies or IL-20RA, IL-20RBor IL-20RA/IL-20RB soluble receptors, or other antagonists such asantibodies against IL-20 or their receptors.

For pharmaceutical use, the soluble polypeptides and antibodies of thepresent invention are formulated for parenteral, particularlyintravenous or subcutaneous, delivery according to conventional methods.Intravenous administration will be by bolus injection, controlledrelease, e.g, using mini-pumps or other appropriate technology, or byinfusion over a typical period of one to several hours. In general,pharmaceutical formulations will include a hematopoietic protein incombination with a pharmaceutically acceptable vehicle, such as saline,buffered saline, 5% dextrose in water or the like. Formulations mayfurther include one or more excipients, preservatives, solubilizers,buffering agents, albumin to prevent protein loss on vial surfaces, etc.When utilizing such a combination therapy, the cytokines may be combinedin a single formulation or may be administered in separate formulations.Methods of formulation are well known in the art and are disclosed, forexample, in Remington's Pharmaceutical Sciences, Gennaro, ed., MackPublishing Co., Easton Pa., 1990, which is incorporated herein byreference. Therapeutic doses will generally be in the range of 0.1 to100 mg/kg of patient weight per day, preferably 0.5-20 mg/kg per day,with the exact dose determined by the clinician according to acceptedstandards, taking into account the nature and severity of the conditionto be treated, patient traits, etc. Determination of dose is within thelevel of ordinary skill in the art. The proteins will commonly beadministered over a period of up to 28 days following chemotherapy orbone-marrow transplant or until a platelet count of >20,000/mm³,preferably >50,000/mm³, is achieved. More commonly, the proteins will beadministered over one week or less, often over a period of one to threedays. In general, a therapeutically effective amount of soluble IL-20RA,IL-20RB or IL-20RA/IL-20RB or anti-IL-20, anti-IL-20RA or anti-IL-20RBantibodies of the present invention is an amount sufficient to produce aclinically significant increase in the proliferation and/ordifferentiation of lymphoid or myeloid progenitor cells, which will bemanifested as an increase in circulating levels of mature cells (e.g.platelets or neutrophils). Treatment of platelet disorders will thus becontinued until a platelet count of at least 20,000/mm³, preferably50,000/mm³, is reached. The soluble IL-20RA, IL-20RB or IL-20RA/IL-20RBor anti-IL-20, anti-IL-20RA or anti-IL-20RB antibodies of the presentinvention can also be administered in combination with other cytokinessuch as IL-3, -6 and -11; stem cell factor; erythropoietin; G-CSF andGM-CSF. Within regimens of combination therapy, daily doses of othercytokines will in general be: EPO, 150 U/kg; GM-CSF, 5-15 lg/kg; IL-3,1-5 lg/kg; and G-CSF, 1-25 lg/kg. Combination therapy with EPO, forexample, is indicated in anemic patients with low EPO levels.

Generally, the dosage of administered soluble IL-20RA, IL-20RB orIL-20RA/IL-20RB or anti-IL-20, anti-IL-20RA or anti-IL-20RB antibodieswill vary depending upon such factors as the patient's age, weight,height, sex, general medical condition and previous medical history.Typically, it is desirable to provide the recipient with a dosage ofsoluble IL-20RA, IL-20RB or IL-20RA/IL-20RB or anti-IL-20, anti-IL-20RAor anti-IL-20RB antibodies which is in the range of from about 1 pg/kgto 10 mg/kg (amount of agent/body weight of patient), although a loweror higher dosage also may be administered as circumstances dictate.

Administration of soluble IL-20RA, IL-20RB or IL-20RA/IL-20RB oranti-IL-20, anti-IL-20RA or anti-IL-20RB antibodies to a subject can beintravenous, intra-arterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, by perfusion through a regionalcatheter, or by direct intralesional injection. When administeringtherapeutic proteins by injection, the administration may be bycontinuous infusion or by single or multiple boluses.

Additional routes of administration include oral, mucosal-membrane,pulmonary, and transcutaneous. Oral delivery is suitable for polyestermicrospheres, zein microspheres, proteinoid microspheres,polycyanoacrylate microspheres, and lipid-based systems (see, forexample, DiBase and Morrel, “Oral Delivery of MicroencapsulatedProteins,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 255-288 (Plenum Press 1997)). The feasibility of anintranasal delivery is exemplified by such a mode of insulinadministration (see, for example, Hinchcliffe and Illum, Adv. DrugDeliv. Rev. 35:199 (1999)). Dry or liquid particles comprising IL-20RAcan be prepared and inhaled with the aid of dry-powder dispersers,liquid aerosol generators, or nebulizers (e.g., Pettit and Gombotz,TIBTECH 16:343 (1998); Patton et al., Adv. Drug Deliv. Rev. 35:235(1999)). This approach is illustrated by the AERX diabetes managementsystem, which is a hand-held electronic inhaler that deliversaerosolized insulin into the lungs. Studies have shown that proteins aslarge as 48,000 kDa have been delivered across skin at therapeuticconcentrations with the aid of low-frequency ultrasound, whichillustrates the feasibility of trascutaneous administration (Mitragotriet al., Science 269:850 (1995)). Transdermal delivery usingelectroporation provides another means to administer a polypeptide ofthe present invention.

A pharmaceutical composition comprising a soluble IL-20RA, IL-20RB orIL-20RA/IL-20RB or anti-IL-20, anti-IL-20RA or anti-IL-20RB antibody canbe formulated according to known methods to prepare pharmaceuticallyuseful compositions, whereby the therapeutic proteins are combined in amixture with a pharmaceutically acceptable carrier. A composition issaid to be a “pharmaceutically acceptable carrier” if its administrationcan be tolerated by a recipient patient. Sterile phosphate-bufferedsaline is one example of a pharmaceutically acceptable carrier. Othersuitable carriers are well-known to those in the art. See, for example,Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (MackPublishing Company 1995).

For purposes of therapy, soluble IL-20RA, IL-20RB or IL-20RA/IL-20RB oranti-IL-20, anti-IL-20RA or anti-IL-20RB antibody molecules and apharmaceutically acceptable carrier are administered to a patient in atherapeutically effective amount. A combination of a therapeuticmolecule of the present invention and a pharmaceutically acceptablecarrier is said to be administered in a “therapeutically effectiveamount” if the amount administered is physiologically significant. Anagent is physiologically significant if its presence results in adetectable change in the physiology of a recipient patient. For example,an agent used to treat inflammation is physiologically significant ifits presence alleviates the inflammatory response.

A pharmaceutical composition comprising the polypeptides of the presentinvention can be furnished in liquid form, in an aerosol, or in solidform. Liquid forms, are illustrated by injectable solutions and oralsuspensions. Exemplary solid forms include capsules, tablets, andcontrolled-release forms. The latter form is illustrated by miniosmoticpumps and implants (Bremer et al., Pharm. Biotechnol. 10:239 (1997);Ranade, “Implants in Drug Delivery,” in Drug Delivery Systems, Ranadeand Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer et al.,“Protein Delivery with Infusion Pumps,” in Protein Delivery: PhysicalSystems, Sanders and Hendren (eds.), pages 239-254 (Plenum Press 1997);Yewey et al., “Delivery of Proteins from a Controlled Release InjectableImplant,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 93-117 (Plenum Press 1997)).

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci. 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta 1068:133 (1991); Allen et al., Biochim. Biophys. Acta1150:9 (1993)).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For example, liposomes, prepared with a high contentof a nonionic surfactant, have been used to target the liver (Hayakawaet al., Japanese Patent 04-244,018; Kato et al., Biol. Pharm. Bull.16:960 (1993)). These formulations were prepared by mixing soybeanphospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull. 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies, antibody fragments, carbohydrates,vitamins, and transport proteins. For example, liposomes can be modifiedwith branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev.Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm.Bull.20:259 (1997)). Similarly, Wu et al., Hepatology 27:772 (1998),have shown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull.20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al., Adv. Drug Deliv.Rev. 32:99 (1998)).

Polypeptides and antibodies can be encapsulated within liposomes usingstandard techniques of protein microencapsulation (see, for example,Anderson et al., Infect. Immun. 31:1099 (1981), Anderson et al., CancerRes. 50:1853 (1990), and Cohen et al., Biochim. Biophys. Acta 1063:95(1991), Alving et al. “Preparation and Use of Liposomes in ImmunologicalStudies,” in Liposome Technology, 2nd Edition, Vol. III, Gregoriadis(ed.), page 317 (CRC Press 1993), Wassef et al., Meth. Enzymol. 149:124(1987)). As noted above, therapeutically useful liposomes may contain avariety of components. For example, liposomes may comprise lipidderivatives of poly(ethylene glycol) (Allen et al., Biochim. Biophys.Acta 1150:9 (1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

As an illustration, pharmaceutical compositions may be supplied as a kitcomprising a container that comprises a polypeptide with a IL-20RA orIL-20RB extracellular domain, e.g., IL-20RA or IL-20RB monomeric,homodimeric, heterodimeric or multimeric soluble receptors, or a IL-20or IL-20RA or IL-20RB antagonist (e.g., an antibody or antibody fragmentthat binds a IL-20RA, IL-20RB or IL-20RA/IL-20RB polypeptide, orneutralizing anti-IL-20, anti-IL-20RA or anti-IL-20RB antibody).Therapeutic polypeptides can be provided in the form of an injectablesolution for single or multiple doses, or as a sterile powder that willbe reconstituted before injection. Alternatively, such a kit can includea dry-powder disperser, liquid aerosol generator, or nebulizer foradministration of a therapeutic polypeptide. Such a kit may furthercomprise written information on indications and usage of thepharmaceutical composition. Moreover, such information may include astatement that the IL-20RA, IL-20RB or IL-20RA/IL-20RB composition iscontraindicated in patients with known hypersensitivity to IL-20RA,IL-20RB or IL-20RA/IL-20RB.

A pharmaceutical composition comprising anti-IL-20, anti-IL-20RA oranti-IL-20RB antibodies or binding partners (or anti-IL-20, anti-IL-20RAor anti-IL-20RB antibody fragments, antibody fusions, humanizedantibodies and the like), or IL-20RA, IL-20RB or IL-20RA/IL-20RB solublereceptor, can be furnished in liquid form, in an aerosol, or in solidform. Liquid forms, are illustrated by injectable solutions, aerosols,droplets, topological solutions and oral suspensions. Exemplary solidforms include capsules, tablets, and controlled-release forms. Thelatter form is illustrated by miniosmotic pumps and implants (Bremer etal., Pharm. Biotechnol. 10:239 (1997); Ranade, “Implants in DrugDelivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.), pages95-123 (CRC Press 1995); Bremer et al., “Protein Delivery with InfusionPumps,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 239-254 (Plenum Press 1997); Yewey et al., “Delivery ofProteins from a Controlled Release Injectable Implant,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 93-117(Plenum Press 1997)). Other solid forms include creams, pastes, othertopological applications, and the like.

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci. 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta 1068:133 (1991); Allen et al., Biochim. Biophys. Acta1150:9 (1993)).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For example, liposomes, prepared with a high contentof a nonionic surfactant, have been used to target the liver (Hayakawaet al., Japanese Patent 04-244,018; Kato et al., Biol. Pharm. Bull.16:960 (1993)). These formulations were prepared by mixing soybeanphospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull. 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies, antibody fragments, carbohydrates,vitamins, and transport proteins. For example, liposomes can be modifiedwith branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev.Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm.Bull. 20:259 (1997)). Similarly, Wu et al., Hepatology 27:772 (1998),have shown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull. 20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al., Adv. Drug Deliv.Rev. 32:99 (1998)).

Anti-IL-20, anti-IL-20RA or anti-IL-20RB neutralizing antibodies andbinding partners with IL-20 binding activity, or IL-20RA, IL-20RB orIL-20RA/IL-20RB soluble receptor, can be encapsulated within liposomesusing standard techniques of protein microencapsulation (see, forexample, Anderson et al., Infect. Immun. 31:1099 (1981), Anderson etal., Cancer Res. 50:1853 (1990), and Cohen et al., Biochim. Biophys.Acta 1063:95 (1991), Alving et al. “Preparation and Use of Liposomes inImmunological Studies,” in Liposome Technology, 2nd Edition, Vol. III,Gregoriadis (ed.), page 317 (CRC Press 1993), Wassef et al., Meth.Enzymol. 149:124 (1987)). As noted above, therapeutically usefulliposomes may contain a variety of components. For example, liposomesmay comprise lipid derivatives of poly(ethylene glycol) (Allen et al.,Biochim. Biophys. Acta 1150:9 (1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

The present invention contemplates compositions of soluble IL-20RA,IL-20RB or IL-20RA/IL-20RB or anti-IL-20, anti-IL-20RA or anti-WL-20RB,and methods and therapeutic uses comprising an such antibodies, peptidesor polypeptides described herein. Such compositions can further comprisea carrier. The carrier can be a conventional organic or inorganiccarrier. Examples of carriers include water, buffer solution, alcohol,propylene glycol, macrogol, sesame oil, corn oil, and the like.

10. Production of Transgenic Mice

Over expression of IL-20 was shown in human psoriatic lesions,suggesting that IL-20 is involved in human psoriasis. Moreover, asdescribed herein, over expression of IL-20 in transgenic mice showedepidermal thickening and immune cell involvement indicative of apsoriatic phenotype. As such, antagonists to IL-20 activity, such as theanti-human-IL-20, anti-human-IL-20RA and anti-human-IL-20RB neutralizingand monoclonal antibodies of the present invention, as well as solubleIL-20RA, IL-20RB or IL-20RA/IL-20RB receptors, are useful in therapeutictreatment of inflammatory-diseases, particularly as antagonists to IL-20in the treatment of psoriasis. Moreover, antagonists to IL-20 activity,such as the anti-human-IL-20, anti-human-IL-20RA and anti-human-IL-20RBneutralizing and monoclonal antibodies of the present invention, as wellas soluble IL-20RA, IL-20RB and IL-20RA/IL-20RB receptors, are useful intherapeutic treatment of other inflammatory diseases for example asantagonists to IL-20 in the treatment of atopic dermatitis, IBD,colitis, Endotoxemia, arthritis, rheumatoid arthritis, and psoriaticarthritis adult respiratory disease (ARD), septic shock, multiple organfailure, inflammatory lung injury such as asthma or bronchitis,bacterial pneumonia, psoriasis, eczema, atopic and contact dermatitis,and inflammatory bowel disease such as ulcerative colitis and Crohn'sdisease, and the like.

Within one aspect, the present invention provides a method of producingan antibody to a polypeptide comprising: inoculating an animal with apolypeptide selected from the group consisting of: (a) a polypeptideconsisting of the amino acid sequence of SEQ ID NO:3 from amino acidnumber 1 (Pro), to amino acid number 6 (Asp); (b) a polypeptideconsisting of the amino acid sequence of SEQ ID NO:3 from amino acidnumber 26 (Ser), to amino acid number 32 (Pro); (c) a polypeptideconsisting of the amino acid sequence of SEQ ID NO:3 from amino acidnumber 41 (Lys), to amino acid number 47 (Asp); (d) a polypeptideconsisting of the amino acid sequence of SEQ ID NO:2 from amino acidnumber 49 (Val), to amino acid number 62 (Cys); (e) a polypeptideconsisting of the amino acid sequence of SEQ ID NO:3 from amino acidnumber 41 (Lys) to amino acid number 62 (Cys); (f) a polypeptideconsisting of the amino acid sequence of SEQ ID NO:3 from amino acidnumber 84 (Ala) to amino acid number 97 (Ser); (g) a polypeptideconsisting of the amino acid sequence of SEQ ID NO:3 from amino acidnumber 103 (Thr) to amino acid number 108 (Asp); (h) a polypeptideconsisting of the amino acid sequence of SEQ ID NO:3 from amino acidnumber 130 (Arg) to amino acid number 135 (His); (i) a polypeptideconsisting of the amino acid sequence of SEQ ID NO:3 from amino acidnumber 164 (Gly) to amino acid number 166 (Lys); (j) a polypeptideconsisting of the amino acid sequence of SEQ ID NO:3 from amino acidnumber 175 (Tyr), to amino acid number 179 (Glu); (k) a polypeptideconsisting of the amino acid sequence of SEQ ID NO:3 from amino acidnumber 193 (Lys) to amino acid number 196 (Ala); (1) a polypeptideconsisting of the amino acid sequence of SEQ ID NO:3 from amino acidnumber 203 (Lys) to amino acid number 209 (Thr); and (m) a polypeptideconsisting of the amino acid sequence of SEQ ID NO:3; and (n) apolypeptide consisting of the amino acid sequence of SEQ ID NO:4; andwherein the polypeptide elicits an immune response in the animal toproduce the antibody; and isolating the antibody from the animal; andwherein the antibody specifically binds to an IL-20RA polypeptide (SEQID NO:2 or SEQ ID NO:3); and inhibits the pro-inflammatory activity ofIL-20 (SEQ ID NO:8) or IL-22 (SEQ ID NO:6).

Within one embodiment is provided the method as described above, whereinthe antibody produced by the method inhibits the pro-inflammatoryactivity of IL-20.

Within a second aspect, the present invention provides an antibodyproduced by the method as disclosed above, which binds to a polypeptideof SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:21,or SEQ ID NO:23. Within one embodiment is provided the antibody asdescribed above, wherein the antibody is selected from the groupconsisting of: (a) a polyclonal antibody, (b) a murine monoclonalantibody, (c) a humanized antibody derived from (b), (d) an antibodyfragment, and (e) a human monoclonal antibody.

Within a third aspect, the present invention provides an antibody orantibody fragment that binds to a polypeptide comprising a sequence ofamino acid residues as shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:21, or SEQ ID NO:23; and inhibits thepro-inflammatory activity of IL-20. Within one embodiment is providedthe antibody as described above, wherein the antibody further comprisesa radionuclide, enzyme, substrate, cofactor, fluorescent marker,chemiluminescent marker, peptide tag, magnetic particle, drug, or toxin.

Within another aspect, the present invention provides a method forinhibiting IL-20-induced proliferation or differentiation ofhematopoietic cells and hematopoietic cell progenitors comprisingculturing bone marrow or peripheral blood cells with a compositioncomprising an amount of an antibody as disclosed above sufficient toreduce proliferation or differentiation of the hematopoietic cells inthe bone marrow or peripheral blood cells as compared to bone marrow orperipheral blood cells cultured in the absence of soluble cytokinereceptor. Within one embodiment is provided the method as describedabove, wherein the hematopoietic cells and hematopoietic progenitorcells are lymphoid cells.

Within another embodiment is provided the method as described above,wherein the lymphoid cells are macrophages or T cells.

Within another aspect, the present invention provides a method ofreducing IL-20-induced inflammation comprising administering to a mammalwith inflammation an amount of a composition of an antibody as disclosedabove sufficient to reduce inflammation.

Within another aspect, the present invention provides a method ofsuppressing an inflammatory response in a mammal with inflammationcomprising: (1) determining a level of serum amyloid A protein; (2)administering a composition comprising an antibody as disclosed above inan acceptable pharmaceutical vehicle; (3) determining a postadministration level of serum amyloid A protein; (4) comparing the levelof serum amyloid A protein in step (1) to the level of serum amyloid Aprotein in step (3), wherein a lack of increase or a decrease in serumamyloid A protein level is indicative of suppressing an inflammatoryresponse.

Within one embodiment is provided the antibody as described above,wherein the antibody further comprises a radionuclide, enzyme,substrate, cofactor, fluorescent marker, chemiluminescent marker,peptide tag, magnetic particle, drug, or toxin.

Within another aspect, the present invention provides a method forinhibiting IL-20-induced proliferation or differentiation ofhematopoietic cells and hematopoietic cell progenitors comprisingculturing bone marrow or peripheral blood cells with a compositioncomprising an amount of an antibody as disclosed above sufficient toreduce proliferation or differentiation of the hematopoietic cells inthe bone marrow or peripheral blood cells as compared to bone marrow orperipheral blood cells cultured in the absence of soluble cytokinereceptor. Within one embodiment is provided the method as describedabove, wherein the hematopoietic cells and hematopoietic progenitorcells are lymphoid cells.

Within another embodiment is provided the method as described above,wherein the lymphoid cells are macrophages or T cells.

Within another aspect, the present invention provides a method ofreducing IL-20-induced inflammation comprising administering to a mammalwith inflammation an amount of a composition of a an antibody asdisclosed above sufficient to reduce inflammation.

Within another aspect, the present invention provides a method ofsuppressing an inflammatory response in a mammal with inflammationcomprising: (1) determining a level of serum amyloid A protein; (2)administering a composition comprising an antibody as disclosed above inan acceptable pharmaceutical vehicle; (3) determining a postadministration level of serum amyloid A protein; (4) comparing the levelof serum amyloid A protein in step (1) to the level of serum amyloid Aprotein in step (3), wherein a lack of increase or a decrease in serumamyloid A protein level is indicative of suppressing an inflammatoryresponse. Within another aspect, the present invention provides a methodof treating a mammal afflicted with an inflammatory disease in whichIL-20 plays a role, comprising: administering an antagonist of IL-20 tothe mammal such that the inflammation is reduced, wherein the antagonistis selected from the group consisting of an antibody or bindingpolypeptide that specifically binds a polypeptide or polypeptidefragment of IL-20, IL-20RA or IL-20RB or is a polypeptide or polypeptidefragment of IL-20, IL-20RA or IL-20RB; and wherein the inflammatoryactivity of IL-20 is reduced. Within one embodiment is provided themethod as described above, wherein the disease is a chronic inflammatorydisease.

Within another embodiment is provided the method as described above,wherein the disease is a chronic inflammatory disease selected from thegroup consisting of: inflammatory bowel disease; ulcerative colitis;Crohn's disease; arthritis; and psoriasis.

Within another embodiment is provided the method as described above,wherein the disease is an acute inflammatory disease. Within anotherembodiment is provided the method as described above, wherein thedisease is an acute inflammatory disease selected from the groupconsisting of: endotoxemia; septicemia; toxic shock syndrome; andinfectious disease.

Within another embodiment is provided the method as described above,wherein the antibody further comprises a radionuclide, enzyme,substrate, cofactor, fluorescent marker, chemiluminescent marker,peptide tag, magnetic particle, drug, or toxin.

Within another aspect, the present invention provides an antibodycomprising a monoclonal antibody that that binds to an epitope of humanIL-20 (SEQ ID NO:2), wherein the epitope comprises a polypeptide havingan amino acid sequence selected from the group consisting of: amino acidresidues 42 (Ile) to 102 (Asp) of SEQ ID NO:2; amino acid residues 42(Ile) to 60 (Ile) of SEQ ID NO:2; amino acid residues 42 (Ile) to 69(Glu) of SEQ ID NO:2; amino acid residues 42 (Ile) to 81 (Cys) of SEQ IDNO:2; amino acid residues 42 (Ile) to 96 (Lys) of SEQ ID NO:2; aminoacid residues 42 (Ile) to 102 (Asp) of SEQ ID NO:2; amino acid residues60 (Ile) to 69 (Glu) of SEQ ID NO:2; amino acid residues 60 (Ile) to 81(Cys) of SEQ ID NO:2; amino acid residues 60 (Ile) to 96 (Lys) of SEQ IDNO:2; amino acid residues 60 (Ile) to 102 (Asp) of SEQ ID NO:2; aminoacid residues 69 (Glu) to 81 (Cys) of SEQ ID NO:2; amino acid residues69 (Glu) to 96 (Lys) of SEQ ID NO:2; amino acid residues 69 (Glu) to 102(Asp) of SEQ ID NO:2; amino acid residues 81 (Cys) to 96 (Lys) of SEQ IDNO:2; amino acid residues 81 (Cys) to 102 (Asp) of SEQ ID NO:2; aminoacid residues 96 (Lys) to 102 (Asp) of SEQ ID NO:2, and wherein theantibody reduces or neutralizes the pro-inflammatory activity of humanIL-20 (SEQ ID NO:2). Within one embodiment is provided the antibody asdescribed above, wherein the antibody further comprises a radionuclide,enzyme, substrate, cofactor, fluorescent marker, chemiluminescentmarker, peptide tag, magnetic particle, drug, or toxin.

Within another embodiment is provided the antibody as described above,wherein the antibody is selected from the group consisting of: (a) amurine monoclonal antibody, (b) a humanized antibody derived from (a),(c) an antibody fragment, and (d) a human monoclonal antibody.

Within another aspect, the present invention provides an antibodycomprising a monoclonal antibody that that binds to an epitope of humanIL-20RA (SEQ ID NO:14), wherein the epitope comprises a polypeptidehaving an amino acid sequence selected from the group consisting of:amino acid residues 1 (Met) to 9 (Leu) of SEQ ID NO:14; amino acidresidues 1 (Met) to 36 (Gly) of SEQ ID NO:14; amino acid residues 1(Met) to 41 (Ala) of SEQ ID NO:14; amino acid residues 1 (Met) to 58(Pro) of SEQ ID NO:14; amino acid residues 1 (Met) to 63 (Gln) of SEQ IDNO:14; amino acid residues 1 (Met) to 80 (Lys) of SEQ ID NO:14; aminoacid residues 1 (Met) to 94 (Tyr) of SEQ ID NO:14; amino acid residues 1(Met) to 104 (Tyr) of SEQ ID NO:14; amino acid residues 1 (Met) to 120(Cys) of SEQ ID NO:14; amino acid residues 1 (Met) to 128 (Arg) of SEQID NO:14; amino acid residues 1 (Met) to 161 (Trp) of SEQ ID NO:14;amino acid residues 1 (Met) to 169 (Pro) of SEQ ID NO:14; amino acidresidues 1 (Met) to 187 (Asn) of SEQ ID NO:14; amino acid residues 1(Met) to 194 (Trp) of SEQ ID NO:14; amino acid residues 1 (Met) to224(Gly) of SEQ ID NO:14; amino acid residues 1 (Met) to 233 (Glu) ofSEQ ID NO:14; amino acid residues 1 (Met) to 316 (Ile) of SEQ ID NO:14;amino acid residues 1 (Met) to 323 (Ile) of SEQ ID NO:14; amino acidresidues 1 (Met) to 335 (Asp) of SEQ ID NO:14; amino acid residues 1(Met) to 340 (Asn) of SEQ ID NO:14; amino acid residues 1 (Met) to 354(Glu) of SEQ ID NO:14; amino acid residues 1 (Met) to 371 (Cys) of SEQID NO:14; amino acid residues 1 (Met) to381 (Ser) of SEQ ID NO:14; aminoacid residues I (Met) to 384 (Gln) of SEQ ID NO:14; amino acid residues1 (Met) to 397 (Thr) of SEQ ID NO:14; amino acid residues 1 (Met) to 412(Ala) of SEQ ID NO:14; amino acid residues 1 (Met) to 418 (Glu) of SEQID NO:14; amino acid residues 1 (Met) to 462 (Gln) of SEQ ID NO:14,amino acid residues 1 (Met) to 476 (Ser) of SEQ ID NO:14; amino acidresidues 1 (Met) to 483 (Asp) of SEQ ID NO:14; amino acid residues 1(Met) to 486 (Thr) of SEQ ID NO:14; amino acid residues 1 (Met) to 496(Ser) of SEQ ID NO:14; amino acid residues 1 (Met) to 511 (Gly) of SEQID NO:14; amino acid residues 1 (Met) to 523 (Glu) of SEQ ID NO:14;amino acid residues 1 (Met) to 536 (Thr) of SEQ ID NO:14; amino acidresidues 36 (Gly) to 63(Gln) of SEQ ID NO:14; amino acid residues 36(Gly) to 94 (tyr) of SEQ ID NO:14; amino acid residues 36 (Gly) to 128(Arg) of SEQ ID NO:14; amino acid residues 36 (Gly) to 169 (Pro) of SEQID NO:14; amino acid residues 36 (Gly) to 194 (Trp) of SEQ ID NO:14;amino acid residues 36 (Gly) to 233 (Glu) of SEQ ID NO:14; amino acidresidues 36 (Gly) to 323 (Ser) of SEQ ID NO:14; amino acid residues 36(Gly) to 340 (Asn) of SEQ ID NO:14; amino acid residues 36 (Gly) to 354(Glu) of SEQ ID NO:14; amino acid residues 36 (Gly) to 381 (Ser) of SEQID NO:14; amino acid residues 36 (Gly) to 397 (Thr) of SEQ ID NO:14;amino acid residues 36 (Gly) to 418 (Glu) of SEQ ID NO:14; amino acidresidues 36 (Gly) to 476 (Ser) of SEQ ID NO:14; amino acid residues 36(Gly) to 486 (Thr) of SEQ ID NO:14; amino acid residues 36 (Gly) to 511(Gly) of SEQ ID NO:14; amino acid residues 36 (Gly) to 536 (Thr) of SEQID NO:14; amino acid residues 58 (Pro) to 63 (Gln) of SEQ ID NO:14;amino acid residues 58 (Pro) to 94 (tyr) of SEQ ID NO:14; amino acidresidues 58 (Pro) to 128 (Arg) of SEQ ID NO:14; amino acid residues 58(Pro) to 169 (Pro) of SEQ ID NO:14; amino acid residues 58 (Pro) to 194(Trp) of SEQ ID NO:14; amino acid residues 58 (Pro) to 233 (Glu) of SEQID NO:14; amino acid residues 58 (Pro) to 323 (Ser) of SEQ ID NO:14;amino acid residues 58 (Pro) to 340 (Asn) of SEQ ID NO:14; amino acidresidues 58 (Pro) to 354 (Glu) of SEQ ID NO:14; amino acid residues 58(Pro) to 381 (Ser) of SEQ ID NO:14; amino acid residues 58 (Pro) to 397(Thr) of SEQ ID NO:14; amino acid residues 58 (Pro) to 418 (Glu) of SEQID NO:14; amino acid residues 58 (Pro) to 476 (Ser) of SEQ ID NO:14;amino acid residues 58 (Pro) to 486 (Thr) of SEQ ID NO:14; amino acidresidues 58 (Pro) to 511 (Gly) of SEQ ID NO:14; amino acid residues 58(Pro) to 536 (Thr) of SEQ ID NO:14; amino acid residues 80 (Lys) to 94(tyr) of SEQ ID NO:14; amino acid residues 80 (Lys) to 128 (Arg) of SEQID NO:14; amino acid residues 80 (Lys) to 169 (Pro) of SEQ ID NO:14;amino acid residues 80 (Lys) to 194 (Trp) of SEQ ID NO:14; amino acidresidues 80 (Lys) to 233 (Glu) of SEQ ID NO:14; amino acid residues 80(Lys) to 323 (Ser) of SEQ ID NO:14; amino acid residues 80 (Lys) to 340(Asn) of SEQ ID NO:14; amino acid residues 80 (Lys) to 354 (Glu) of SEQID NO:14; amino acid residues 80 (Lys) to 381 (Ser) of SEQ ID NO:14;amino acid residues 80 (Lys) to 397 (Thr) of SEQ ID NO:14; amino acidresidues 80 (Lys) to 418 (Glu) of SEQ ID NO:14; amino acid residues 80(Lys) to 476 (Ser) of SEQ ID NO:14; amino acid residues 80 (Lys) to 486(Thr) of SEQ ID NO:14; amino acid residues 80 (Lys) to 511 (Gly) of SEQID NO:14; amino acid residues 80 (Lys) to 536 (Thr) of SEQ ID NO:14;amino acid residues 120 (Cys) to 128 (Arg) of SEQ ID NO:14; amino acidresidues 120 (Cys) to 169 (Pro) of SEQ ID NO:14; amino acid residues 120(Cys) to 194 (Trp) of SEQ ID NO:14; amino acid residues 120 (Cys) to 233(Glu) of SEQ ID NO:14; amino acid residues 120 (Cys) to 323 (Ser) of SEQID NO:14; amino acid residues 120 (Cys) to 340 (Asn) of SEQ ID NO:14;amino acid residues 120 (Cys) to 354 (Glu) of SEQ ID, NO:14; amino acidresidues 120 (Cys) to 381 (Ser) of SEQ ID NO:14; amino acid residues 120(Cys) to 397 (Thr) of SEQ ID NO:14; amino acid residues 120 (Cys) to 418(Glu) of SEQ ID NO:14; amino acid residues 120 (Cys) to 476 (Ser) of SEQif) NO:14; amino acid residues 120 (Cys) to 486 (Thr) of SEQ ID NO:14;amino acid residues 120 (Cys) to 511 (Gly) of SEQ ID NO:14; amino acidresidues 120 (Cys) to 536 (Thr) of SEQ ID NO:14; amino acid residues 161(Trp) to 169 (Pro) of SEQ ID NO:14; amino acid residues 161 (Trp) to 194(Trp) of SEQ ID NO:14; amino acid residues 161 (Trp) to 233 (Glu) of SEQID NO:14; amino acid residues 161 (Trp) to 323 (Ser) of SEQ ID NO:14;amino acid residues 161 (Trp) to 340 (Asn) of SEQ ID NO:14; amino acidresidues 161 (Trp) to 354 (Glu) of SEQ ID NO:14; amino acid residues 161(Trp) to 381 (Ser) of SEQ ID NO:14; amino acid residues 161 (Trp) to 397(Thr) of SEQ ID NO:14; amino acid residues 161 (Trp) to 418 (Glu) of SEQID NO:14; amino acid residues 161 (Trp) to 476 (Ser) of SEQ ID NO:14;amino acid residues 161 (Trp) to 486 (Thr) of SEQ ID NO:14; amino acidresidues 161 (Trp) to 511 (Gly) of SEQ ID NO:14; amino acid residues 161(Trp) to 536 (Thr) of SEQ ID NO:14; amino acid residues 187 (Asn) to 194(Trp) of SEQ ID NO:14; amino acid residues 187 (Asn) to 233 (Glu) of SEQID NO:14; amino acid residues 187 (Asn) to 323 (Ser) of SEQ ID NO:14;amino acid residues 187 (Asn) to 340 (Asn) of SEQ ID NO:14; amino acidresidues 187 (Asn) to 354 (Glu) of SEQ ID NO:14; amino acid residues 187(Asn) to 381 (Ser) of SEQ ID NO:14; amino acid residues 187 (Asn) to 397(Thr) of SEQ ID NO:14; amino acid residues 187 (Asn) to 418 (Glu) of SEQID NO:14; amino acid residues 187 (Asn) to 476 (Ser) of SEQ ID NO:14;amino acid residues 187 (Asn) to 486 (Thr) of SEQ ID NO:14; amino acidresidues 187 (Asn) to 511 (Gly) of SEQ ID NO:14; amino acid residues 187(Asn) to 536 (Thr) of SEQ ID NO:14; amino acid residues 224 (Gly) to 233(Glu) of SEQ ID NO:14; amino acid residues 224 (Gly) to 323 (Ser) of SEQID NO:14; amino acid residues 224 (Gly) to 340 (Asn) of SEQ ID NO:14;amino acid residues 224 (Gly) to 354 (Glu) of SEQ ID NO:14; amino acidresidues 224 (Gly) to 381 (Ser) of SEQ ID NO:14; amino acid residues 224(Gly) to 397 (Thr) of SEQ ID NO:14; amino acid residues 224 (Gly) to 418(Glu) of SEQ ID NO:14; amino acid residues 224 (Gly) to 476 (Ser) of SEQID NO:14; amino acid residues 224 (Gly) to 486 (Thr) of SEQ ID NO:14;amino acid residues 224 (Gly) to 511 (Gly) of SEQ ID NO:14; amino acidresidues 224 (Gly) to 536 (Thr) of SEQ ID NO:14; amino acid residues 316(Ile) to 323 (Ser) of SEQ ID NO:14; amino acid residues 316 (Ile) to 340(Asn) of SEQ ID NO:14; amino acid residues 316 (Ile) to 354 (Glu) of SEQID NO:14; amino acid residues 316 (Ile) to 381 (Ser) of SEQ ID NO:14;amino acid residues 316 (Ile) to 397 (Thr) of SEQ ID NO:14; amino acidresidues 316 (Ile) to 418 (Glu) of SEQ ID NO:14; amino acid residues 316(Ile) to 476 (Ser) of SEQ ID NO:14; amino acid residues 316 (Ile) to 486(Thr) of SEQ ID NO:14; amino acid residues 316 (Ile) to 511 (Gly) of SEQID NO:14; amino acid residues 316 (Ile) to 536 (Thr) of SEQ ID NO:14;amino acid residues 335 (Asp) to 340 (Asn) of SEQ ID NO:14; amino acidresidues 335 (Asp) to 354 (Glu) of SEQ ID NO:14; amino acid residues 335(Asp) to 381 (Ser) of SEQ ID NO:14; amino acid residues 335 (Asp) to 397(Thr) of SEQ ID NO:14; amino acid residues 335 (Asp) to 418 (Glu) of SEQID NO:14; amino acid residues 335 (Asp) to 476 (Ser) of SEQ ID NO:14;amino acid residues 335 (Asp) to 486 (Thr) of SEQ ID NO:14; amino acidresidues 335 (Asp) to 511 (Gly) of SEQ ID NO:14; amino acid residues 335(Asp) to 536 (Thr) of SEQ ID NO:14; amino acid residues 371 (Cys) to 381(Ser) of SEQ ID NO:14; amino acid residues 371 (Cys) to 397 (Thr) of SEQID NO:14; amino acid residues 371 (Cys) to 418 (Glu) of SEQ ID NO:14;amino acid residues 371 (Cys) to 476 (Ser) of SEQ ID NO:14; amino acidresidues 371 (Cys) to 486 (Thr) of SEQ ID NO:14; amino acid residues 371(Cys) to 511 (Gly) of SEQ ID NO:14; amino acid residues 371 (Cys) to 536(Thr) of SEQ ID NO:14; amino acid residues 384 (Gln) to 397 (Thr) of SEQID NO:14; amino acid residues 384 (Gln) to 418 (Glu) of SEQ ID NO:14;amino acid residues 384 (Gin) to 476 (Ser) of SEQ ID NO:14; amino acidresidues 384 (Gin) to 486 (Thr) of SEQ ID NO:14; amino acid residues 384(Gln) to 511 (Gly) of SEQ ID NO:14; amino acid residues 384 (Gln) to 536(Thr) of SEQ ID NO:14; amino acid residues 412 (Ala) to 418 (Glu) of SEQID NO:14; amino acid residues 412 (Ala) to 476 (Ser) of SEQ ID NO:14;amino acid residues 412 (Ala) to 486 (Thr) of SEQ ID NO:14; amino acidresidues 412 (Ala) to 511 (Gly) of SEQ ID NO:14; amino acid residues 412(Ala) to 536 (Thr) of SEQ ID NO:14; amino acid residues 462 (Gln) to 476(Ser) of SEQ ID NO:14; amino acid residues 462 (Gln) to 486 (Thr) of SEQID NO:14; amino acid residues 462 (Gln) to 511 (Gly) of SEQ ID NO:14;amino acid residues 462 (Gln) to 536 (Thr) of SEQ ID NO:14; amino acidresidues 483 (Asp) to 486 (Thr) of SEQ ID NO:14; amino acid residues 483(Asp) to 511 (Gly) of SEQ ID NO:14; amino acid residues 483 (Asp) to 536(Thr) of SEQ ID NO:14; amino acid residues 496 (Ser) to 511 (Gly) of SEQID NO:14; amino acid residues 496 (Ser) to 536 (Thr) of SEQ ID NO:14;amino acid residues 523 (Glu) to 536 (Thr) of SEQ ID NO:14, and whereinthe antibody reduces or neutralizes the pro-inflammatory activity ofhuman IL-20 (SEQ ID NO:2). Within one embodiment is provided theantibody as described above, wherein the antibody further comprises aradionuclide, enzyme, substrate, cofactor, fluorescent marker,chemiluminescent marker, peptide tag, magnetic particle, drug, or toxin.

Within another embodiment is provided the antibody as described above,wherein the antibody is selected from the group consisting of: (a) amurine monoclonal antibody, (b) a humanized antibody derived from (a),(c) an antibody fragment, and (d) a human monoclonal antibody.

Within another aspect, the present invention provides an antibodycomprising a monoclonal antibody that that binds to an epitope of humanIL-20RB (SEQ ID NO:21), wherein the epitope comprises a polypeptidehaving an amino acid sequence selected from the group consisting of:amino acid residues 70 (Tyr) to 74 (Tyr) of SEQ ID NO:21; amino acidresidues 70 (Tyr) to 101 (Asp) of SEQ ID NO:21; amino acid residues 70(Tyr) to 135 (Ser) of SEQ ID NO:21; amino acid residues 70 (Tyr) to 178(Glu) of SEQ ID NO:21; amino acid residues 70 (Tyr) to 283 (Lys) of SEQID NO:21; amino acid residues 92 (Thr) to 101 (Asp) of SEQ ID NO:21;amino acid residues 92 (Thr) to 135 (Ser) of SEQ ID NO:21; amino acidresidues 92 (Thr) to 178 (Glu) of SEQ ID NO:21; amino acid residues 92(Thr) to 283 (Lys) of SEQ ID NO:21; amino acid residues 130 (Pro) to 135(Ser) of SEQ ID NO:21; amino acid residues 130 (Pro) to 178 (Glu) of SEQID NO:21; amino acid residues 130 (Pro) to 283 (Lys) of SEQ ID NO:21;amino acid residues 171 (Arg) to 178 (Glu) of SEQ ID NO:21; amino acidresidues 171 (Arg) to 283 (Lys) of SEQ ID NO:21; amino acid residues 279(Asn) to 283 (Lys) of SEQ ID NO:21, and wherein the antibody reduces orneutralizes the pro-inflammatory activity of human IL-20 (SEQ ID NO:2).Within one embodiment is provided the antibody as described above,wherein the antibody further comprises a radionuclide, enzyme,substrate, cofactor, fluorescent marker, chemiluminescent marker,peptide tag, magnetic particle, drug, or toxin.

Within another embodiment is provided the antibody as described above,wherein the antibody is selected from the group consisting of: (a) amurine monoclonal antibody, (b) a humanized antibody derived from (a),(c) an antibody fragment, and (d) a human monoclonal antibody.

Within another aspect, the present invention provides a method oftreating a pathological condition in a subject associated with IL-20activity comprising administering an effective amount of the antibody asdisclosed above thereby treating said pathological condition. Within oneembodiment is provided the method as described above, wherein saidpathological condition is a chronic inflammatory condition.

Within another embodiment is provided the method as described above,wherein said chronic inflammatory condition is selected from the groupconsisting of: inflammatory bowel disease; ulcerative colitis; Crohn'sdisease; arthritis; and psoriasis. Within another embodiment is providedthe method as described above, wherein said pathological condition is anacute inflammatory condition.

Within another embodiment is provided the method as described above,wherein said acute inflammatory condition is selected from the groupconsisting of: endotoxemia; septicemia; toxic shock syndrome; andinfectious disease.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLE 1 Cloning of IL-20

Cloning of Human IL-20

The full length sequence of IL-20×1 (the longer form—SEQ ID NO:1) andIL-20×2 (the shorter form—SEQ ID NO:4) was elucidated by using 3′ RACE®and submitting two fragments generated to sequencing (SEQ ID NO:30 andSEQ ID NO:31), then artificially splicing together by computer the estsequence shown in SEQ ID NO:32 with the overlapping sequence from thetwo 3′ race fragments.

An oligo, zc15907 (SEQ ID NO:33), was designed to the area just upstream(5′) of the putative methionine for IL-20. Further downstream, anotheroligo, zc15906 (SEQ ID NO: 34), was designed to the area just upstreamof the signal sequence cleavage site. These oligos were used in 3′ RACEreactions on human trachea marathon cDNA. ZC15907 was used in theprimary 3′ race reaction and zc15906 was used in the nested 3′ racereaction. The MARATHON cDNA was made using the Marathon cDNAAmplification Kit (Clontech, Palo Alto, Calif.) according to themanufacturer's instructions, starting with human trachea mRNA purchasedfrom Clontech.

The PCR reactions were run according to the manufacturer's instructionsin the Marathon cDNA Amplification Kit with some modification in thethermal cycling parameters. The cycling parameters used in the primaryPCR reaction were:

-   -   94° C. 1 min 30sec 1×    -   94° C. 15 sec 68° C. 1 min 30×    -   72° C. 7 min 1×

The cycling parameters used in the nested PCR reaction were: 94° C. 1min 30 sec 1×, 94° C. 15 sec 68° C. 1 min 20 sec, 30×72° C. 7 min 1×.

The resulting products were run out on a 1.2% agarose gel (Gibcoagarose) and two main bands were seen, approximately 80 bp apart. Thebands were cut out and gel purified using QIAEX™ resin (Qiagen)according to the manufacturer's instructions. These fragments were thensubjected to sequencing, allowing the full length sequence of IL-20 tobe discerned.

Cloning of Murine IL-20

PCR primers 5′ MARATHON RACE™ (Clontech, Palo Alto, Calif.) primer setSEQ ID NO: 35 attached to MARATHON™ AP1 adapter, nested with SEQ IDNO:36 attached to AP2 MARATHON™ adapter, with 3′ MARATHON RACE™ primerset SEQ ID NO: 37 attached to MARATHON RACE™ AP1 adapter, nested withSEQ ID NO:38 attached to MARATHON RACE™ AP2 adapter and 5′ and 3′ racewas performed on mouse skin MARATHON RACE™ cDNA. Several fragments werefrom these reactions were gel purified and sequenced, allowing theelucidation of the full length coding sequence of the mouse IL-20, plussome 5′ and 3′ UTR sequence. Two murine IL-20 variants were discovered,namely SEQ ID NOs: 39 and 40 and SEQ ID NOs: 41 and 42. The clones wereamplified by PCR using primers SEQ ID NOs:43 and 44.

EXAMPLE 2 Cloning of IL-20RA

Cloning of Human IL-20RA

Expressed sequence tag (EST) 277139 (SEQ ID NO:45) was identified. ThecDNA clone (ID No. 50416) was obtained from the IMAGE consortiumLawrence Livermore National Laboratory through Genome Systems, Inc. ThecDNA was supplied as an agar stab containing E. coli transfected with aplasmid having the cDNA of interest. The E. coli was streaked on an agarplate. The plasmid was designated pSL7139. The cDNA insert in plasmidpSL7139 was sequenced. The insert was determined to be 1231 bp inlength, but was not a full length sequence.

A human testis cDNA template was made using a MARATHON™ cDNAAmplification Kit (Clontech Laboratories, Inc., Palo Alto, Calif.)according to the supplier's instructions. A 5

RACE reaction was used to obtain a full-length cDNA. The RACE reactionwas carried out in two reactions employing two sets of primers. ReactionI (outer nest), using primers ZC11,107 (SEQ ID NO:46) and AP-1 (SEQ IDNO: 47) (Clontech Laboratories) was run for 35 cycles at 98° C. for 20seconds, 45° C. for 20 seconds; 68° for 4 minutes and a final extensiontime of 10 minutes at 68° C. One μl of a 1:100 dilution of the reactionproduct was used as a template in reaction II (inner nest). Primers wereZC11,108 (SEQ ID NO:48 ) and AP-2 (SEQ ID NO:49 ) (ClontechLaboratories). The reaction was run at 98° C. for 30 seconds, and 30cycles each cycle being comprised of 98° C. for 28 seconds; 43° C. for20 seconds; and 68° C. for 3.5 minutes with a final extension at 68° C.for 10 minutes.

The product of the inner nest RACE reaction was subcloned using aPCR-SCRIPT™ kit (Stratagene Cloning Systems, La Jolla, Calif.) toprepare the plasmid pSLR7-1. Sequence analysis of this plasmid indicatedthat the 5

RACE-generated sequence extended the sequence of pSL7139 by 555 bp.

Full-length cDNA was obtained by screening a λZAP® II human testis cDNAlibrary using a probe that was generated by PCR primers ZC11,526 (SEQ IDNO:50) and ZC11,108 (SEQ ID NO:48) and pSLR7-1 as template and thenre-amplified. The resulting probe was purified through recovery fromlow-melt agarose gel electrophoresis and was labeled with ³²P-α-dCTPusing a MEGAPRIME™ labeling kit (Amersham Corp., Arlington, Heights,Ill.). The labeled probe was purified on a push column (NUCTRAP® probepurification column; Stratagene Cloning Systems).

The first strand cDNA reaction contained 15 μl of human testis twicepoly d(T)-selected poly (A)⁺ mRNA (Clontech Laboratories) at aconcentration of 1.0 μg/l, and 3 μl of 20 pmole/μl first strand primerZC6091 (SEQ ID NO:51) containing an Xho I restriction site. The mixturewas heated at 70° C. for 4 minutes and cooled by chilling on ice. Firststand cDNA synthesis was initiated by the addition of 12 μl of firststrand buffer (5× SUPERSCRIP™ buffer; Life Technologies, Gaithersburgh,Md.), 6 μl of 100 mM dithiothreitol, 3 μl of deoxynucleotidetriphosphate solution containing 10 mM each of dTTP, dATP, dGTP, and5-methyl-dCTP (Pharmacia LKB Biotechnology, Piscataway, N.J.) to theRNA-primer mixture. The reaction mixture was incubated at 37° C. for 2minutes, followed by the addition of 15 μl of 200 U/μl Rnase H⁻ reversetranscriptase (SUPERSCRIPT II^(<<); Life Technologies). The efficiencyof the first strand synthesis was analyzed in a parallel reaction by theaddition of 5 μCi of ³²P-αdCTP to 5 g aliquot from one of the reactionmixtures to label the reaction for analysis. The reactions wereincubated at 37° C. for 10 minutes, 45° C. for 1 hour, then incubated at50° C. for 10 minutes. Unincorporated ³²P-αdCTP in the labeled reactionand the unincorporated nucleotides and primers in the unlabeled firststrand reactions were removed by chromatography on a 400 pore size gelfiltration column (Clontech Laboratories). The length of labeled firststrand cDNA was determined by agarose gel electrophoresis.

The second strand reaction contained 120 μl of the unlabeled firststrand cDNA, 36 μl of 5× polymerase 1 buffer (125 mM Tris: HCl, pH 7.5,500 mM KCl, 25 mM MgCl₂,50 mM (NH₄)₂SO₄)), 2.4 μl of 100 mMdithiothreitol, 3.6 μl of a solution containing 10 mM of eachdeoxynucleotide triphosphate, 6 μl of 5 mM β-NAD, 3.6 μl of 3 U/μl E.coli DNA ligase (New England Biolabs),9 μl of 10 U/μl E. coli DNApolymerase I (New England Biolabs), and 1.8 μl of 2 U/μl RNase H (lifeTechnologies). A 10 μl aliquot from one of the second strand synthesisreactions was labeled by the addition of 10 μCi ³²P-αdCTP to monitor theefficiency of second strand synthesis. The reactions were incubated at16° C. for two hours, followed by the addition of 15 μl T4 DNApolymerase (10 U/μl, Boerhinger Mannheim, Indianapolis, Ind.) andincubated for an additional 5 minutes at 16° C. Unincorporated ³²P-αdCTPin the labeled reaction was removed by chromatography through a 400 poresize gel filtration (Clontech Laboratories) before analysis by agarosegel electrophoresis. The unlabeled second strand reaction was terminatedby the addition of 20 μl 0.5 M EDTA and extraction withphenol/chloroform and chloroform followed by ethanol precipitation inthe presence of 2.5 M ammonium acetate and 4 μg of glycogen carrier. Theyield of cDNA was estimated to be approximately 3 μg from starting mRNAtemplate of 15 μg.

Eco RI adapters were ligated onto the 5

ends of the cDNA described above to enable cloning into an expressionvector. A 10 μl aliquot of cDNA (approximately 1.5 μg) and 5 μl of 65pmole/μl of Eco RI adapter (Pharmacia LKB Biotechnology Inc.) were mixedwith 2 μl 10× ligase buffer (660 mM Tris-HCl pH 7.5, 100 mM MgCl₂), 2 μlof 10 mM ATP and 1 μl of 15 U/μl T4 DNA ligase (Promega Corp., Madison,Wis.). The reaction was incubated 2 hours at 5° C., two hours at 7.5°C., 2 hours at 10° C., and 10 hours at 12.5° C. The reaction wasterminated by incubation at 70° C. for 20 minutes.

To facilitate the directional cloning of the cDNA into an expressionvector, the cDNA was digested with Xho I, resulting in a cDNA having a 5

Eco RI cohesive end and a 3

Xho cohesive end. The Xho I restriction site at the 3

end of the cDNA had been previously introduced using the ZC6091 primer(SEQ ID NO: 51). Restriction enzyme digestion was carried out in areaction mixture containing 20 μl of cDNA as described above, 10 μl of10× H Buffer Xho I (Boehringer Mannheim), 69 μl H₂O,and 1.0 μl of 40U/μl Xho I (Boehringer Mannheim). Digestion was carried out at 37° C.for 40 minutes. The reaction was terminated by incubation at 70° C. for10 minutes and chromatography through a 400 pore size gel filtrationcolumn (Clontech Laboratories).

The cDNA was ethanol precipitated, washed with 70% ethanol, air driedand resuspended in 14 μl water, 2 μl of ligase buffer (Promega Corp.,Madison, Wis.), 2 μl T4 polynucleotide kinase (10 U/μl, LifeTechnologies). Following incubation at 37° C. for 30 minutes, the cDNAwas heated to 65° C. for 5 minutes, cooled on ice, and electrophoresedon a 0.8% low melt agarose gel. The contaminating adapters and cDNAbelow 0.6 kb in length were excised from the gel. The electrodes werereversed, and the cDNA was electrophoresed until concentrated near thelane origin. The area of the gel containing the concentrated cDNA wasexcised and placed in a microfuge tube, and the approximate volume ofthe gel slice was determined. An aliquot of water approximately threetimes the volume of the gel slice (300 μl) and 35 μl 10× β-agarose Ibuffer (New England Biolabs) were added to the tube, and the agarose wasmelted by heating to 65° C. for 15 minutes. Following equilibration ofthe sample to 45° C., 3 μl of 1 U/μl β-agarose I (New England Biolabs)was added, and the mixture was incubated for 60 minutes at 45° C. todigest the agarose. After incubation, 40 μl of 3 M Na acetate was addedto the sample, and the mixture was incubated on ice for 15 minutes. Thesample was centrifuged at 14,000×g for 15 minutes at room temperature toremove undigested agarose. The cDNA was ethanol precipitated, washed in70% ethanol, air-dried and resuspended in 10 μl water.

The resulting cDNA was cloned into the lambda phage vector λZap^(<<) II(Stratagene Cloning Systems) that was predigested with Eco RI and Xho Iand dephosphorylated. Ligation of the cDNA to the λZap<< II vector wascarried out in a reaction mixture containing 1.0 μl of prepared vector,1.0 μl of human testis cDNA, 1.0 μl 10× Ligase Buffer (Promega Corp.),1.0 μl of 10 mM ATP, 5 μl water, and 1.0 μl of T4 DNA Ligase at 15units/ml (Promega Corp.). The ligation mixture was incubated at 5°-15°C. overnight in a temperature gradient. After incubation, the ligationmixture was packaged into phage using an in vitro packaging extract(Gigapack<< II Gold packaging extract; Stratagene Cloning Systems), andthe resulting library was titered according to the manufacturer'sspecifications.

The human testis λZAP<< II library was used to infect E. coli host cells(XL1-Blue MRF

strain (Stratagene Cloning Systems), and 1.5×10⁶ plaque forming units(pfu) were plated onto 150-mm NZY plates at a density of about 50,000pfu/plate. The inoculated plates were incubated overnight at 37° C.Filter plaque lifts were made using nylon membranes (Hybond™-N; AmershamCorp., Arlington Heights, Ill.), according to the procedures provided bythe manufacturer. The filters were processed by denaturation in solutioncontaining 1.5 M NaCl and 0.5 M NaOH for 6 minutes at room temperature.The filters were blotted briefly on filter paper to remove excessdenaturation solution, followed by neutralization for 6 minutes in 1 MTris-HCl, pH 7.5, and 1.5 M NaCl. Phage DNA was fixed onto the filterswith 1,200 μJoules of UV energy in a UV Crosslinker (Stratalinker<<;Stratagene Cloning Systems). After fixing, the filters were firstpre-washed in an aqueous solution containing 0.25× standard sodiumcitrate (SSC), 0.25% sodium dodecyl sulfate (SDS) and imM EDTA to removecellular debris and then prehybridized in hybridization solution (5×SSC,5× Denhardt's solution, 0.2% SDS and 1 mM DTA). Heat-denatured, shearedsalmon sperm DNA at a final concentration of 100 μg/ml was added. Thefilters were prehybridized at 65° C. overnight.

A probe was prepared as a PCR product by using oligonucleotide primersdesigned to amplify the human IL-20RA coding region. A PCR reactionmixture was prepared containing 2 μl of ZC11526 (SEQ ID NO:50) 2 μl ofZC11,108 (SEQ ID NO:48), 1 μl of an overnight bacterial culture ofpSLR7-1, 1 μl of 10 mM dNTP, 10 μl of 10× KlenTaq buffer (ClontechLaboratories), 82 μl water, and 2 μl KlenTaq DNA polymerase (Clontechlaboratories). The PCR reaction was run as follows: 94° C. for 1 minute;30 cycles of 95° C. for 20 seconds, 43° C. for 20 seconds, 68° C. for 1minute; then held at 68° C. for 10 minutes. The PCR product wasre-amplified and gel purified on a 0.8% low melt agarose gel.

Fifty nanograms PCR product was radiolabeled with ³²P-α-dCTP by randompriming using the MEGAPRIME<< DNA Labeling System (Amersham), accordingto the manufacturer's specifications. The prehybridization solution wasreplaced with fresh hybridization solution containing 1.4×10⁶ cpm/mllabeled probe and allowed to hybridize for 64 hours at 60° C. Afterhybridization, the hybridization solution was removed and the filterswere rinsed in a wash solution containing 0.25×SSC, 0.25% SDS and 1 mMEDTA at 65° C. The filters were placed on autoradiograph film andexposed at −70° C. with intensifying screens for 72 hours.

Examination of the autoradiographs revealed multiple regions thathybridized with labeled probe. Agar plugs were picked from 12 regionsfor purification. Each agar plug was soaked 2 hours in 0.5 ml of SMsolution containing 25 ml 4M NaCl, 10 ml 1M MgSO₄, 25 ml 2M Tris HCl, 5ml 2% gelatin and 935 ml H₂O and 10% (v/v) chloroform (Sambrook et al.Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989). After incubation, thephages from each plug were diluted 1:1000 in SM. Aliquots of 50 μl wereplated on 100 mm plates containing 300 μl of E. coli XL-1 Blue MRF

cells. The plates were incubated overnight at 37° C., and filter liftswere prepared, prehybridized overnight, hybridized overnight with ahybridization solution containing 1.1×10⁶ cpm/ml labeled probe, washedand autoradiographed. Examination of the resulting autoradiographsrevealed 10 positive signals. The positive plaques were subjected to anadditional round of purification.

The plasmids were excised using an ExASSIST/SOLR<< system (StratageneCloning Systems), according to the manufacturer's specifications. Theseplasmid inserts were amplified by PCR for size determination. A clone,designated pSLR7-2 was sequenced and determined to have an insert of3,532 bp in size.

Cloning of Murine IL-20RA

A cross-species hybridization probe was generated which contained thefull-length cDNA fragment encoding human IL-20RA. A Southern blot ofmouse genomic DNA and Northern blots of mouse RNA were performed todemonstrate that the human IL-20RA cDNA could specifically hybridize tomouse sequences. The Northern blot results indicated that mouse IL-20RARNA was present in mouse embryo day 15 and 17 as well as heart, brain,lung, liver, kidney, testes, spleen, thymus, liver, stomach, and smallintestine.

The human IL-20RA full length DNA hybridization probe was used to screena mouse genomic library. The library, which was obtained from Clontech(Palo Alto, Calif.), was generated from an MboI partial digest of mousegenomic DNA and cloned into the BamHI site of Lambda bacteriophage EMBL3SP6/T7. Positive bacteriophage was plaque purified and bacteriophage DNAwas prepared using Promega's Wizard Lambda Preps DNA PurificationSystem. Two genomic restriction enzyme fragments, a 5.7 kb EcoRIfragment and an 8.0 kb Sacd fragment, were generated from the positivebacteriophage and subcloned into pBluescript. DNA sequence analysisrevealed the presence of 3 exons from the mouse ortholog to humanIL-20RA.

PCR primers from the 5′ UTR (SEQ ID NO: 52) and 3′ UTR (SEQ ID NO: 53)were designed to generate a full-length mouse IL-20RA sequence by PCRamplification. Mouse embryo 15 day plus 17 day cDNA was used as thetemplate for the PCR amplification. PCR products were subcloned andsequenced for confirmation. The mouse sequences are SEQ ID NOs: 54 and55. The mature extracellular domain is comprised of SEQ ID NO: 56.

EXAMPLE 3 Cloning of Human IL-20RB

Two PCR primers were designed based on the sequence from InternationalPatent Application No. PCT/US99/03735 filed on Mar. 8, 1999. SEQ ID NO:57 contains the ATG (Met1) codon with an EcoRI restriction site, SEQ IDNO: 58 contains the stop codon (TAG) with an XhoI restriction site. ThePCR amplification was carried out using a human keratinocyte (HaCaT)cDNA library DNA as a template and SEQ ID NO: 59 and SEQ ID NO: 58 asprimers. The PCR reaction was performed as follows: incubation at 94° C.for 1 min followed by 30 cycles of 94° C. for 30 sec and 68° C. for 2min, after additional 68° C. for 4 min, the reaction was stored at 4° C.The PCR products were run on 1% Agarose gel, and a 1 kb DNA band wasobserved. The PCR products were cut from the gel and the DNA waspurified using a QIAquick Gel Extraction Kit (Qiagen). The purified DNAwas digested with EcoRI and XhoI, and cloned into a pZP vector that wascalled pZP7N. A pZP plasmid is a mammalian expression vector containingan expression cassette having the mouse metallothionein-1 promoter,human tPA leader peptide, multiple restriction sites for insertion ofcoding sequences, a Glu-Glu tag, and a human growth hormone terminator.The plasmid also has an E. coli origin of replication, a mammalianselectable marker expression unit having an SV40 promoter, an enhancerand an origin of replication, as well as a DHFR gene, and the SV40terminator. Several IL-20RB-pZP7N clones were sequenced. They allcontain three non-conservative mutations compared with the sequence ofIL-20RB in PCT/US99/03735: (sequence IL-20RB-pZP7N), 146 Pro (CCC)—Thr(ACC), 148 His (CAT)—Asp (GAT), and 171 Thr (ACG)—Arg (AGG).

To verify the three substitutions in IL-20RB-pZP7N clone, PCRamplification was carried out using three difference cDNA sources—fetalskin marathon cDNA, HaCaT cDNA library DNA, and prostate smooth musclecDNA library DNA—as templates. The PCR products were gel purified andsequenced. The sequence of each of the three PCR products was consistentwith that of the IL-20RB-pZP7N clone. IL-20RB is SEQ ID NO: 20 and 21,and the mature extracellular domain is SEQ ID NO: 59.

EXAMPLE 4 IL-20RA/RB Receptor-Ig Fusion Heterotetramer

The expression vector pEZE3 was used to express the recombinant IL-20receptor-Ig fusion protein. The plasmid pEZE3 is derived from pDC312.pDC312 was obtained through license from Immunex Corporation. Theplasmids pDC312 and pEZE3 contain an EASE segment as described in WO97/25420. The presence of the EASE segment in an expression vector canimprove expression of recombinant proteins two to eight fold in stablecell pools.

The plasmid pEZE3 is a tricistronic expression vector that may be usedto express up to three different proteins in mammalian cells, preferablyChinese Hamster Ovary (CHO) cells. The pEZE3 expression unit containsthe cytomegalovirus (CMV) enhancer/promoter, the adenovirus tripartiteleader sequence, a multiple cloning site for insertion of the codingregion for the first recombinant protein, the poliovirus type 2 internalribosome entry site, a second multiple cloning site for insertion of thecoding region for the second recombinant protein, anencephalomyocarditis virus internal ribosome entry site, a codingsegment for mouse dihydrofolate reductase, and the SV40 transcriptionterminator. In addition, pEZE3 contains an E. coli origin of replicationand the bacterial beta lactamase gene.

The IL-20 receptor-Ig fusion protein is a disulfide linkedheterotetramer consisting of two chains of the extracellular domain ofthe human IL-20RB fused to the wild type human immunoglobulin kappalight chain constant region and two chains of the human IL-20RA proteinextracellular domain fused to a mutated human immunoglobulin gamma 1constant region. The human immunoglobulin gamma 1 constant regioncontains amino acid substitutions to reduce FcγRI binding and C1qcomplement fixation.

The human IL-20RB extracellular domain human immunoglobulin kappa lightchain constant region fusion construct was generated by overlap PCR. TheIL-20RB coding segment consists of amino acids 1 to 230 of SEQ ID NO:20. The template used for the PCR amplification of the IL-20R segmentwas generated IL-20RB human kappa light chain constant region expressionconstruct as described below. Oligonucleotide primers SEQ ID NO: 60 andSEQ ID NO: 61 were used to amplify the IL-20RB segment. The entire wildtype human immunoglobulin kappa light chain constant region was used.The template used for the PCR amplification of the wild type humanimmunoglobulin kappa light chain constant region segment was generatedIL-20RB human kappa light chain constant region expression construct asdescribed in Example 12. Oligonucleotide primers SEQ ID NO: 62 and SEQID NO: 63 were used to amplify the wild type human immunoglobulin kappalight chain constant region. The two protein coding domains were linkedby overlap PCR using oligonucleotides SEQ ID NO: 60 and SEQ ID NO: 63. A(Gly₄Ser)₃ (SEQ ID NO: 64) peptide linker was inserted between the twoprotein domains. The (Gly₄Ser)₃ peptide linker was encoded on the PCRprimers SEQ ID NO: 61 and SEQ ID NO:62. The resultant WL-20RBextracellular domain/kappa light chain constant region fusion constructis shown by SEQ ID NOs: 65 and 66. The predicted mature polypeptide,minus the signal sequence, is SEQ ID NO: 67. The portion of theextracellular domain of IL-20RB that was actually used was comprised ofthe amino acid sequence of SEQ ID NO: 26. N-terminal sequencing resultedin the predicted amino acid sequence.

The human IL-20RA extracellular domain human immunoglobulin gamma 1heavy chain constant region fusion construct was generated by overlapPCR of four separate DNA fragments, each generated by separate PCRamplification reactions. The first fragment contained an optimized tPA(tissue plasminogen activator) signal sequence. The tPA signal sequencewas amplified using oligonucleotide primers SEQ ID NO: 68 and SEQ ID NO:69 using an in-house previously generated expression vector as thetemplate. The second fragment contained the IL-20RA extracellulardomain-coding region consisting of amino acids 30 to 243 of SEQ ID NO:14. Oligonucleotide primers SEQ ID NO: 70 and SEQ ID NO: 71 were used toamplify this IL-20RA segment using a previously generated clone ofIL-20RA as the template.

The human gamma 1 heavy chain constant region was generated from 2segments. The first segment containing the C_(H)1 domain was amplifiedusing oligonucleotide primers SEQ ID NO: 72 and SEQ ID NO: 73 using aclone of the wild type human gamma 1 heavy chain constant region as thetemplate. The second segment containing the remaining hinge, C_(H)2, andC_(H)3 domains of the human immunoglobulin gamma 1 heavy chain constantregion was generated by PCR amplification using oligonucleotide primersSEQ ID NO: 74 and SEQ ID NO: 75. The template used for this PCRamplification was from a previously generated human gamma 1 Fc constructthat contained codons for amino acid substitutions to reduce FcγR1binding and C1q complement fixation.

The four protein coding domains were linked by overlap PCR usingoligonucleotides SEQ ID NO: 68 and SEQ ID NO: 75. A (Gly₄Ser)₃ peptidelinker was inserted between the IL-20RA and CH1 protein domains. The(Gly₄Ser)₃ peptide linker was encoded on the PCR primers SEQ ID NO: 71and SEQ ID NO: 72. The IL-20RA extracellular domain/domain humanimmunoglobulin gamma 1 heavy constant region fusion protein and DNAsequence are shown in SEQ ID NOs: 76 and 77. The predicted maturepolypeptide sequence, minus the signal sequence, is SEQ ID NO: 78. Theportion of extracellular domain of IL-20RA that was actually used wascomprised of SEQ ID NO: 79.

The IL-20RB extracellular domain human immunoglobulin kappa light chainconstant region fusion coding segment was cloned into the second MCSwhile the human IL-20RA extracellular domain human immunoglobulin gamma1 heavy chain constant region fusion coding segment was cloned into thefirst MCS of pEZE3. The plasmid was used to transfect CHO cells. Thecells were selected in medium without hypoxanthine or thymidine and thetransgene was amplified using methotrexate. The presence of protein wasassayed by Western blotting using anti human gamma 1 heavy chainconstant region and anti human kappa light chain antibodies. N-terminalsequencing revealed that the optimized tPA leader was not completelycleaved. The observed mass indicated that the first residue of thepolypeptide sequence to be pyroglutamic acid, and the N-terminalsequence appears to be pyroEEIHAELRRFRRVPCVSGG (SEQ ID NO: 80), theunderlined portion being remnants of the tPA leader.

EXAMPLE 5 Construction of an IL-20 Receptor Heterodimer

A vector expressing a secreted human IL-20RA/hIL-20B heterodimer wasconstructed. In this construct, the extracellular domain of humanIL-20RA was fused to the heavy chain of IgG gamma 1 (IgGγ1), while theextracellular portion of IL-20RB was fused to human kappa light chain(human κ light chain).

Construction of IgG Gamma 1 and Human κ Light Fusion Vectors

The heavy chain of IgGγ1 was cloned into the Zem229R mammalianexpression vector (ATCC deposit No. 69447) such that any extracellularportion of a receptor having a 5′ EcoRI and 3′ NheI site can be clonedin, resulting in an N-terminal extracellular domain-C-terminal IgGγ1fusion. The IgGγ1 fragment used in this construct was made by using PCRto isolate the IgGγ1 sequence from a Clontech hFetal Liver cDNA libraryas template. A PCR reaction using oligos (SEQ ID NO: 61) ZC11,450 and(SEQ ID NO: 62) ZC11,443 was run as follows: 40 cycles of 94° C. for 60sec., 53° C. for 60 sec., and 72° C. for 120 sec.; and 72° C. for 7 min.PCR products were separated by agarose gel electrophoresis and purifiedusing a QiaQuick™ (Qiagen) gel extraction kit. The isolated, 990 bp, DNAfragment was digested with MluI and EcoRI (Boerhinger-Mannheim), ethanolprecipitated and ligated with oligos (SEQ ID NO: 63) ZC11,440 and (SEQID NO: 68) ZC11,441, which comprise an MluI/EcoRI linker, into Zem229Rpreviously digested with MluI and EcoRI using standard molecular biologytechniques disclosed herein. This generic cloning vector was calledVector#76 hIgGgamma1 w/ Ch1 #786 Zem229R (Vector #76). Thepolynucleotide sequence of the extracellular domain of hIL-20RA fused tothe heavy chain of IgG gamma 1 is show in SEQ ID NO: 69 and thecorresponding polypeptide sequence shown in SEQ ID NO: 70, the maturesequence of which is SEQ ID NO: 81.

The human κ light chain was cloned in the Zem228R mammalian expressionvector (ATCC deposit No. 69446) such that any extracellular portion of areceptor having a 5′ EcoRI site and a 3′ KpnI site can be cloned in,resulting in an N-terminal extracellular domain-C-terminal human κ lightchain fusion. The human κ light chain fragment used in this constructwas made by using PCR to isolate the human κ light chain sequence fromthe same Clontech hFetal Liver cDNA library used above. A PCR reactionusing oligos (SEQ ID NO: 71) ZC11,501 and (SEQ ID NO: 72) ZC11,451 wasrun under condition described above. PCR products were separated byagarose gel electrophoresis and purified using a QiaQuick™ (Qiagen) gelextraction kit. The isolated, 315 bp, DNA fragment was digested withMluI and EcoRI (Boerhinger-Mannheim), ethanol precipitated and ligatedwith the MluI/EcoRI linker described above, into Zem228R previouslydigested with MluI and EcoRI using standard molecular biology techniquesdisclosed herein. This generic cloning vector was called Vector #77hκlight #774 Zem228R (Vector #77). The polynucleotide sequence of theextracellular portion of IL-20RB fused to human kappa light chain isshown in SEQ ID NO: 73 and the corresponding polypeptide sequence shownin SEQ ID NO: 74, the mature sequence of which is SEQ ID NO: 82.

Insertion of IL-20RA and IL-20RB Extracellular Domains into FusionVector Constructs

Using the construction vectors above, a construct having human IL-20RAfused to IgGγ1 was made. This construction was done by PCRing humanIL-20RA receptor from hIL-20RA/IgG Vector #102 with oligos (SEQ ID NO:75) ZC12,909 and (SEQ ID NO: 83) ZC26,564 under conditions described asfollows: 30 cycles of 94° C. for 60 sec., 57° C. for 60 sec., and 72° C.for 120 sec.; and 72° C. for 7 min. The resulting PCR product wasdigested with EcoRI and NheI, gel purified, as described herein, andligated into a previously EcoRI and NheI digested and band-purifiedVector #76 (above). The resulting vector was sequenced to confirm thatthe human IL-20Rα/IgG gamma 1 fusion (hIL-20RA/Ch1 IgG) was correct. ThehIL-20RA/Ch1 IgG gamma 1 #1825 Zem229R vector was called vector #195.

A separate construct having IL-20RB fused to κ light was alsoconstructed. The IL-20RB/human κ light chain construction was performedas above by PCRing from DR1/7N-4 with oligos (SEQ ID NO: 84) ZC26,602and (SEQ ID NO: 85) ZC26,599, digesting the resulting band with EcoRIand KpnI and then ligating this product into a previously EcoRI and KpnIdigested and band-purified Vec#77 (above). The resulting vector wassequenced to confirm that the IL-20RB/human κ light chain fusion(IL-20RB/κlight) was correct. This IL-20RB/κlight #1833 Zem228R vectorwas called Vector #194.

Co-Expression of the Human IL-20RA and Human IL-20RB Receptors

Approximately 16 μg of each of vectors #194 and #195, above, wereco-transfected into BHK-570 cells (ATCC No. CRL-10314) usingLipofectaminePlus™ reagent (Gibco/BRL), as per manufacturer'sinstructions. The transfected cells were selected for 10 days inDMEM+5%FBS (Gibco/BRL) containing 1 μM of methotrexate (MTX) (Sigma, St.Louis, Mo.) and 0.5 mg/ml G418 (Gibco/BRL) for 10 days. The resultingpool of tranfectants was selected again in 10 μM MTX and 0.5 mg/ml G418for 10 days.

The resulting pool of doubly selected cells was used to generateprotein. Three factories (Nunc, Denmark) of this pool were used togenerate 8 L of serum free conditioned medium. This conditioned mediawas passed over a 1 ml protein-A column and eluted in (10) 750microliter fractions. 4 of these fractions found to have the highestconcentration were pooled and dialyzed (10 kD MW cutoff) against PBS.Finally, the dialyzed material was analyzed by BCA (Pierce) and found tohave a concentration of 317 μg/ml. A total of 951 μg was obtained fromthis 8 L purification.

EXAMPLE 6 Binding of IL-20 to the IL-20RB/IL-20RA Heterodimer

A cell-based binding assay was used to verify IL-20 binds toIL-20RA-IL-20RB heterodimer. Expression vectors containing known andorphan Class II cytokine receptors (including IL-20RA and IL-20RB) weretransiently transfected into COS cells in various combinations, whichwere then assayed for their ability to bind biotin-labeled IL-20protein. The results show IL-20RB- IL-20RA heterodimer is a receptor forIL-20. The procedure used is described below.

The COS cell transfection was performed in a 12-well tissue cultureplate as follows: 0.5 μg DNA was mixed with medium containing 5 μllipofectamine in 92 μl serum free Dulbecco's modified Eagle's medium(DMEM) (55 mg sodium pyruvate, 146 mg L-glutamine, 5 mg transferrin, 2.5mg insulin, 1 μg selenium and 5 mg fetuin in 500 ml DMEM), incubated atroom temperature for 30 minutes and then added to 400 μl serum free DMEMmedia. This 500 μl mixture was then added to 1.5×10⁵ COS cells/well andincubated for 5 hours at 37° C. 500 μl 20% fetal bovine serum (FBS) DMEMmedia was added and incubated overnight.

The assay, a modification of the “secretion trap” (Davis, S., et al.,Cell 87: 1161-1169 (1996), was performed as follows: cells were rinsedwith PBS/1% bovine serum albumin (BSA) and blocked for 1 hour with TNB(0.1 M Tris-HCl, 0.15 M NaCl and 0.5% Blocking Reagent (NEN RenaissanceTSA-Direct Kit Cat# NEL701) in water). This was followed by a one-hourincubation with 3 μg/ml biotinylated IL-20 protein in TNB. Cells werewashed with PBS/1% BSA and incubated for another hour with 1:300 dilutedstreptavidin-HRP (NEN kit) in TNB. Following another wash, cells werefixed for 15 minutes with 1.8% Formaldehyde in phosphate-buffered saline(PBS). Cells were then washed with TNT (0.1 M Tris-HCL, 0.15 M NaCl, and0.05% Tween-20 in water). Positive binding signals were detectedfollowing a five-minute incubation with fluorescein tyramide reagentdiluted 1:50 in dilution buffer (NEN kit). Cells were washed with TNT,preserved with Vectashield Mounting Media (Vector Labs) diluted 1:5 inTNT, and visualized using an FITC filter on an inverted fluorescentmicroscope.

EXAMPLE 7 IL-20RA/RB Receptor-Ig Fusion Heterotetramer

The expression vector pEZE3 was used to express the recombinant IL-20receptor-Ig fusion protein. The plasmid pEZE3 is derived from pDC312.pDC312 was obtained through license from Immunex Corporation. Theplasmids pDC312 and pEZE3 contain an EASE segment as described in WO97/25420. The presence of the EASE segment in an expression vector canimprove expression of recombinant proteins two to eight fold in stablecell pools.

The plasmid pEZE3 is a tricistronic expression vector that may be usedto express up to three different proteins in mammalian cells, preferablyChinese Hamster Ovary (CHO) cells. The pEZE3 expression unit containsthe cytomegalovirus (CMV) enhancer/promoter, the adenovirus tripartiteleader sequence, a multiple cloning site for insertion of the codingregion for the first recombinant protein, the poliovirus type 2 internalribosome entry site, a second multiple cloning site for insertion of thecoding region for the second recombinant protein, anencephalomyocarditis virus internal ribosome entry site, a codingsegment for mouse dihydrofolate reductase, and the SV40 transcriptionterminator. In addition, pEZE3 contains an E. coli origin of replicationand the bacterial beta lactamase gene.

The IL-20 receptor-Ig fusion protein is a disulfide linkedheterotetramer consisting of two chains of the extracellular domain ofthe human IL-20RB fused to the wild type human immunoglobulin kappalight chain constant region and two chains of the human IL-20RA proteinextracellular domain fused to a mutated human immunoglobulin gamma 1constant region. The human immunoglobulin gamma 1 constant regioncontains amino acid substitutions to reduce FcγRI binding and C1qcomplement fixation.

The human IL-20RB extracellular domain human immunoglobulin kappa lightchain constant region fusion construct was generated by overlap PCR. TheIL-20RB coding segment consists of amino acids 1 to 230 of SEQ ID NO:20.The template used for the PCR amplification of the IL-20R segment wasgenerated IL-20RB human kappa light chain constant region expressionconstruct as described herein. Oligonucleotide primers SEQ ID NO: 60 andSEQ ID NO: 61 were used to amplify the IL-20RB segment. The entire wildtype human immunoglobulin kappa light chain constant region was used.The template used for the PCR amplification of the wild type humanimmunoglobulin kappa light chain constant region segment was generatedIL-20RB human kappa light chain constant region expression construct asdescribed in Example 12. Oligonucleotide primers SEQ ID NO: 62 and SEQID NO: 63 were used to amplify the wild type human immunoglobulin kappalight chain constant region. The two protein coding domains were linkedby overlap PCR using oligonucleotides SEQ ID NO: 60 and SEQ ID NO: 63. A(Gly₄Ser)₃ (SEQ ID NO: 64) peptide linker was inserted between the twoprotein domains. The (Gly₄Ser)₃ peptide linker was encoded on the PCRprimers SEQ ID NO: 61 and SEQ ID NO: 62. The resultant IL-20RBextracellular domain/kappa light chain constant region fusion constructis shown by SEQ ID NOs: 65 and 66. The predicted mature polypeptide,minus the signal sequence, is SEQ ID NO: 67. The portion of theextracellular domain of IL-20RB that was actually used was comprised ofthe amino acid sequence of SEQ ID NO: 86. N-terminal sequencing resultedin the predicted amino acid sequence.

The human IL-20RA extracellular domain human immunoglobulin gamma 1heavy chain constant region fusion construct was generated by overlapPCR of four separate DNA fragments, each generated by separate PCRamplification reactions. The first fragment contained an optimized tPA(tissue plasminogen activator) signal sequence. The tPA signal sequencewas amplified using oligonucleotide primers SEQ ID NO: 68 and SEQ ID NO:69 using an in-house previously generated expression vector as thetemplate. The second fragment contained the IL-20RA extracellulardomain-coding region consisting of amino acids 30 to 243 of SEQ ID NO:14. Oligonucleotide primers SEQ ID NO: 70 and SEQ ID NO: 71 were used toamplify this IL-20RA segment using a previously generated clone ofIL-20RA as the template.

The human gamma 1 heavy chain constant region was generated from 2segments. The first segment containing the C_(H)1 domain was amplifiedusing oligonucleotide primers SEQ ID NO: 72 and SEQ ID NO: 73 using aclone of the wild type human gamma 1 heavy chain constant region as thetemplate. The second segment containing the remaining hinge, C_(H)2, andC_(H)3 domains of the human immunoglobulin gamma 1 heavy chain constantregion was generated by PCR amplification using oligonucleotide primersSEQ ID NO: 74 and SEQ ID NO: 75. The template used for this PCRamplification was from a previously generated human gamma 1 Fc constructthat contained codons for amino acid substitutions to reduce FcγRIbinding and C1q complement fixation as described herein.

The four protein coding domains were linked by overlap PCR usingoligonucleotides SEQ ID NO: 68 and SEQ ID NO: 75. A (Gly₄Ser)₃ peptidelinker was inserted between the IL-20RA and CH1 protein domains. The(Gly₄Ser)₃ peptide linker was encoded on the PCR primers SEQ ID NO: 72and SEQ ID NO: 71. The IL-20RA extracellular domain/domain humanimmunoglobulin gamma 1 heavy constant region fusion protein and DNAsequence are shown in SEQ ID NOs: 76 and 77. The predicted maturepolypeptide sequence, minus the signal sequence, is SEQ ID NO: 78. Theportion of extracellular domain of IL-20RA that was actually used wascomprised of SEQ ID NO: 79.

The IL-20RB extracellular domain human immunoglobulin kappa light chainconstant region fusion coding segment was cloned into the second MCSwhile the human IL-20RA extracellular domain human immunoglobulin gamma1 heavy chain constant region fusion coding segment was cloned into thefirst MCS of pEZE3. The plasmid was used to transfect CHO cells. Thecells were selected in medium without hypoxanthine or thymidine and thetransgene was amplified using methotrexate. The presence of protein wasassayed by Western blotting using anti human gamma 1 heavy chainconstant region and anti human kappa light chain antibodies. N-terminalsequencing revealed that the optimized tPA leader was not completelycleaved. The observed mass indicated that the first residue of thepolypeptide sequence to be pyroglutamic acid, and the N-terminalsequence appears to be pyroEEIHAELRRFRRVPCVSGG (SEQ ID NO: 80), theunderlined portion being remnants of the tPA leader.

EXAMPLE 8 Purification of Baculovirus Expressed IL-20RB-TbX-Fc4 FusionProtein and Recovery of the pDIRS 1 Fragment

Purification of IL-20RB-TbX-Fc4 Polypeptide from Baculovirus Insect CellConditioned Media

Unless otherwise noted, all operations were carried out at 4° C., andall liquid chromatography procedures were done via an Applied BiosystemsBioCad workstation (Framingham, Mass.). IL-20RB-TbX-Fc4 fusionpolypeptide was directly captured from the conditioned media via aconjugated Protein A resin available from Applied Biosystems. Thecentrifuged and sterile filtered condition media was adjusted to 0.02%w/v sodium azide and chilled to 4° C., then directly loaded onto anappropriate sized and PBS (Gibco/BRL) equilibrated POROS 50 A column,according to the manufacture's specifications. Captured protein was theneluted from the column with a step gradient of 0.1M glycine pH 3.0.Collected fractions were immediately pH neutralized via a predeterminedvolume of 2M tris pH 8.0 added to the collection tubes, and thefractions of interest were determined via SDS PAGE (Invitrogen,Carlsbad, Calif.) silver staining analysis (Geno Tech. Inc., St. Louis,Mo.). The pooled fractions were sterile filtered and the proteinconcentration was estimated by an UV absorbance reading of 280 nm. Thefinal material was stored at −80° C. until further processing wasinitiated.

In order to remove any high molecular weight aggregates or smallerpolypeptide contaminants, a size exclusion chromatography was performedon the Protein A captured material. The protein A captured pool wasthawed, concentrated against a YM30 30 kD MWCO stirred cell concentratormembrane (Millipore, Bedford, Mass.) to a nominal volume, then loadedonto an appropriately sized Pharmacia sephacryl 200 size exclusioncolumn (Piscataway, N.J.), according to the manufacture'sspecifications. Fractions of interest were determined via SDS PAGEanalysis, pooled, and sterile filtered. The protein concentration wasdetermined via BCA analysis (Pierce, Rockford, Ill.).

Thrombin Activated Enzymatic Digest of the pDIRS-TbX-Fc4 Polypeptide

To separate IL-20RB from the Fc4 portion of the full-length molecule,thrombin was used to promote a sequence specific cleavage at theengineered thrombin cleavage site linker between IL-20RB and Fc4domains. In addition, the full-length polypeptide was immobilized on aprotein A resin prior to the thrombin-activated cleavage to providepurification of IL-20RB from the Fc4. A known amount of the purifiedfull-length polypeptide was added to an appropriate slurry volume of PBSpH 7.2 washed and equilibrated POROS 50 A resin, and allowed to batchabsorb overnight with proper mixing. The batch absorption vial waswarmed to room temperature, then a predetermined amount (1:100 w/w,enzyme to target) of rhthrombin (ZymoGenetics, Inc) was added to thereaction. The enzymatic process was continued for the predetermined timeof 10 minutes at room temperature with proper mixing, and then theslurry was collected into a glass gravity column (Bio-Rad, Hercules,Calif.).

Purification of the Thrombin Liberated IL-20RB Polypeptide Fragment

In order to separate IL-20RB from the thrombin, an ABA resin was used toselectively bind the thrombin out of solution. The eluate and threecolumn volume washes from the post enzymatic reaction slurry werecollected on ice, then equilibrated to 0.5 M NaCl & 20 mM tris pH 8.0.An appropriately sized Tosohass TSK-GEL ABA-5PW Guardgel gravity column(Montgomeryville, Pa.) was washed and equilibrated in 0.5 M NaCl & 20 mMtris pH 8.0, according to the manufacture's specifications. Applicationof the buffer adjusted post enzymatic fractions were applied slowly overthe ABA column, and that column's eluates and washes were also collectedon ice.

To provide a final separation of IL-20RB from any high molecular weightaggregates or smaller polypeptide contaminants, a size exclusionchromatography was performed. This method also provided a bufferexchange in to the formulation buffer of choice. The collected fractionswere pooled then concentrated against a Millipore 5 kD MWCO centrifugalconcentrator to a nominal volume. The concentrate was then applied to aPBS pH 7.2 equilibrated Pharmacia superdex 75 size exclusion column.Fractions of interest were determined via SDS PAGE analysis, pooled,sterile filtered, vialed and stored under proper established conditions.

Characterization of IL-20RB

The final product was characterized by the following methods: SDS PAGEanalyses (including coomassie stain and Western analysis), BCA, AAA, andN-terminal sequencing. SDS PAGE analyses showed a doublet band with agel migration of an approximately 25 kD polypeptide. Both AAA andn-terminal sequencing provided evidence of a sample of high purity and asingle N-terminus.

EXAMPLE 9 Up-Regulation of Inflammatory Cytokines by IL-20

Cell Treatment

The human keratinocyte cell line, HaCaT was grown at 37° C. to severaldays post-confluence in T-75 tissue culture flasks. At this point,normal growth media (DMEM+10% FBS) was removed and replaced withserum-free media. Cells were then incubated for two days at 37° C. DMEMwas then removed and four flasks of cells per treatment were treatedwith one of each of the following conditions for four hours at 37° C.:recombinant human (rh) IL-1 alpha at 5 ng/mL, rh IL-1 alpha at 20 ng/mL,rh IL-1 alpha at 5 ng/mL+IL-20 at 1 μg/n IL-20 at 1 μg/mL, or rh IL-10at 10 ng/mL.

RNA Isolation

Following cytokine treatment, media was removed and cells were lysedusing a guanidium thiocyanate solution. Total RNA was isolated from thecell lysate by an overnight spin on a cesium chloride gradient. Thefollowing day, the RNA pellet was resuspended in a TE/SDS solution andethanol precipitated. RNA was then quantitated using aspectrophotometer, followed by a DNase treatment as per Section V.B. ofClontech's Atlas™ cDNA Expression Arrays User Manual (versionPT3140-1/PR9X390, published Nov. 5, 1999). Quality of RNA samples wasverified by purity calculations based on spec readings, and byvisualization on agarose gel. Genomic contamination of the RNA sampleswas ruled out by PCR analysis of the beta-actin gene.

Probe Synthesis

Clontech's protocols for polyA+ enrichment, probe synthesis andhybridization to Atlas™ arrays were followed (see above, plus Atlas™Pure Total RNA Labeling System User Manual, PT3231-1/PR96157, publishedJun. 22, 1999). Briefly, polyA+ RNA was isolated from 50 mg of total RNAusing streptavidin coated magnetic beads (by Clontech, Paolo Alto,Calif.) and a magnetic particle separator. PolyA+ RNA was then labeledwith ^(alpha32)P-dATP via RT-PCR. Clontech CDS primers specific to the268 genes on the Atlas™ human cytokine/receptor array (Cat. #7744-1)were used in the reaction. Labeled probe was isolated using columnchromatography and counted in scintillation fluid.

Array Membrane Hybridization

Atlas™ arrays were pre-hybridized with Clontech ExpressHyb plus 100mg/mL heat denatured salmon sperm DNA for at least thirty minutes at 68°C. with continuous agitation. Membranes were then hybridized with1.9×10⁶ CPM/mL (a total of 1.14×10⁷ CPM) overnight at 68° C. withcontinuous agitation. The following day, membranes were washed forthirty minutes×4 in 2×SSC, 1% SDS at 680 C, plus for thirty minutes×1 in0.1×SSC, 0.5% SDS at 68° C., followed by one final room temperature washfor five minutes in 2×SSC. Array membranes were then placed in Kodakplastic pouches sealed and exposed to a phosphor imager screen overnightat room temperature. The next day, phosphor screens were scanned on aphosphor imager and analyzed using Clontech's AtlasImage™ 1.0 software.

Genes Up-Regulated by IL-20

-   -   1. Tumor necrosis factor (TNF) was up-regulated 1.9-2.4 fold by        IL-20.    -   2. Placental growth factors 1 & 2 (PLGF) were up-regulated        1.9-2.0 fold by IL-20.    -   3. Coagulating factor HI receptor was up-regulated 2.0-2.5 fold        by IL-20.    -   4. Calcitonin receptor was up-regulated 2.2-2.3 fold by IL-20.    -   5. TNF-inducible hyaluronate-binding protein TSG-6 was        up-regulated 2.1-2.2 fold by IL-20.    -   6. Vascular endothelial growth factor (VEGF) receptor-1        precursor, tyrosine-protein kinase receptor (FLT-1) (SFLT) was        up-regulated 2.1-2.7 fold by IL-20.    -   7. MRP-8 (calcium binding protein in macrophages MIF-related)        was up-regulated 2.9-4.1 fold by IL-20.    -   8. MRP-14 (calcium binding protein in macrophages MIF-related)        was up-regulated 3.0-3.8 fold by IL-20.    -   9. Relaxin H2 was up-regulated 3.14 fold by IL-20.    -   10. Transforming growth factor beta (TGFβ) receptor III 300 kDa        was up-regulated 2.4-3.6 fold by IL-20.        Genes Showing Synergy with IL-20+IL-1 Treatment    -   1. Bone morphogenic protein 2a was up-regulated 1.8 fold with        IL-20 treatment alone, 2.5 fold with IL-1 treatment alone, and        8.2 fold with both IL-20 and IL-1 treatment together.    -   2. MRP-8 was up-regulated 2.9 fold with IL-20 treatment alone,        10.7 fold with IL-1 treatment alone and 18.0 fold with both        IL-20 and IL-1 treatment together.    -   3. Erythroid differentiation protein (EDF) was up-regulated 1.9        fold with IL-20 treatment alone, 9.7 fold with IL-1 treatment        alone and 19.0 fold with both IL-20 and IL-1 treatment together.    -   4. MRP-14 (calcium binding protein in macrophages, MN related)        was up-regulated 3.0 fold with IL-20 treatment alone, 12.2 fold        with IL-1 treatment alone and 20.3 fold with both IL-20 and IL-1        treatment together.    -   5. Heparin-binding EGF-like growth factor was up-regulated 2.0        fold with IL-20 treatment alone, 14 fold with IL-1 treatment        alone and 25.0 fold with both IL-20 and IL-1 treatment together.    -   6. Beta-thromboglobulin-like protein was up-regulated 1.5 fold        with IL-20 treatment alone, 15 fold with IL-1 treatment alone        and 27 fold with both IL-20 and IL-1 treatment together.    -   7. Brain-derived neurotrophic factor (BDNF) was up-regulated 1.7        fold with IL-20 treatment alone, 25 fold with IL-1 treatment        alone and 48 fold with both IL-20 and IL-1 treatment together.    -   8. Monocyte chemotactic and activating factor MCAF was        up-regulated 1.3 fold with IL-20 treatment alone, 32 fold with        IL-1 treatment alone and 56 fold with both IL-20 and IL-1        treatment together.

EXAMPLE 10 Specificity and Affinity of IL-20 for its Receptor

The specificity and affinity of IL-20 for its receptor was determinedusing BHK cells stably transfected with IL-20RA, IL-20RB or bothreceptor subunits. Binding assays using radiolabeled ligand demonstratedthat IL-20 bound to BHK transfectants expressing both IL-20RA andIL-20RB but not to untransfected cells nor to transfectants expressingeither receptor subunit alone. Binding of ¹²⁵I-labeled IL-20 waseliminated in the presence of 100-fold excess of unlabeled IL-20 but notwith 100-fold excess of the unrelated cytokine, IL-21. The bindingaffinity (kD) of IL-20 to the IL-20RA/IL-20RB heterodimeric receptor wasdetermined to be approximately 1.5 nM.

EXAMPLE 11 IL-20 Receptor Activation

To determine if IL-20 binding leads to receptor activation, thefactor-dependent pre-B cell line BaF3 was co-transfected with IL-20RAand IL-20RB and treated with IL-20 at various concentrations. IL-20stimulated proliferation in a dose-dependent manner and gave adetectable signal at 1.1 pM, with a half maximal response at 3.4 pM. Wenote that the IL-20 concentration for the half maximal proliferativeresponse in BaF3 cells is 1000× lower than that for half maximal bindingaffinity in BHK cells. Possible explanations for this large differenceinclude the use of different cell lines, different receptor expressionlevels and different assay outputs. IL-20 also stimulated signaltransduction in the biologically relevant human keratinocyte cell lineHaCaT, which naturally expresses IL-20RA and IL-20RB. Therefore, IL-20binds and activates the heterodimeric IL-20RA/IL-20RB receptor atconcentrations expected for a cytokine. While the negative controlscontaining untransfected BaF3.

EXAMPLE 12 Expression Analysis of IL-20RA and IL-20RB

RT-PCR analysis was performed on a variety of human tissues to determinethe expression pattern of IL-20RA and IL-20RB. Both receptor subunitsare most highly expressed in skin and testis. The significant result isthat IL-20RA and IL-20RB are both expressed in skin, where they havebeen shown to mediate the IL-20-induced response. Both IL-20RA andIL-20RB are also both expressed in monocytes, lung, ovary, muscle,testis, adrenal gland, heart, salivary gland and placenta. XL-20RA isalso in brain, kidney, liver, colon, small intestine, stomach, thyroid,pancreas, uterus and prostate while IL-20RB is not.

EXAMPLE 13 IL-20 is Upregulated in Human Psoriatic Skin Samples

RNA Samples

Normal skin samples as well as skin from psoriasis patients wereobtained. The latter included involved skin from psoriasis and fromadjacent uninvolved skin. RNA was isolated from human skin samples usingconventional methods. The integrity and quality of RNA samples wastested on the Agilent 2100 Bioanalyzer (Agilent Technologies, WaldbronnGermany).

Primers and Probes for Quantitative RT-PCR-

Real-time quantitative RT-PCR using the ABI PRISM 7700 SequenceDetection System (PE Applied Biosystems, Inc., Foster City, Calif.) hasbeen previously described (See, Heid, C. A. et al., Genome Research6:986-994, 1996; Gibson, U. E. M. et al., Genome Research 6:995-1001,1996; Sundaresan, S. et al., Endocrinology 139:4756-4764, 1998. Thismethod incorporates use of a gene specific probe containing bothreporter and quencher fluorescent dyes. When the probe is intact thereporter dye emission is negated due to the close proximity of thequencher dye. During PCR extension using additional gene-specificforward and reverse primers, the probe is cleaved by the 5′ to 3′nucleolytic activity of the rTth DNA Polymerase which releases thereporter dye from the probe resulting in an increase in fluorescentemission.

The primers and probes used for real-time quantitative RT-PCR analysesof IL-20 expression were designed using the primer design softwarePrimer Express™ (PE Applied Biosystems, Foster City, Calif.). Theforward primer, ZC40541 (SEQ ID NO:87) and the reverse primer, ZC 40542(SEQ ID NO:88) were used in a PCR reaction (below) at a 800 nMconcentration to synthesize a 71 bp product. The corresponding IL-20TaqMan® probe, ZC 40544 (SEQ ID NO:89) was synthesized and labeled by PEApplied Biosystems. The IL-20 probe was labeled at the 5′ end with areporter fluorescent dye (6-carboxy-fluorescein) (FAM) (PE AppliedBiosystems) and at the 3′ end with a quencher fluorescent dye(6-carboxy-tetramethyl-rhodamine) (TAMRA) (PE Applied Biosystems).

Real-Time Quantitative RT-PCR

Relative levels of IL-20 mRNA were determined by analyzing total RNAsamples using the TaqMan EZ RT-PCR Core Reagents Kit (PE AppliedBiosystems). Runoff IL-20 transcript was made to generate a standardcurve used for quantitation. The curve consisted of 10-fold serialdilutions ranging from 1e8 to 1e3 total copies of whole message forIL-20 with each standard curve point analyzed in triplicate. The totalRNA samples from skin were also analyzed in triplicate for human IL-20transcript levels and for levels of hGUS as an endogenous control. In atotal volume of 25 μl, each RNA sample was subjected to TaqMan EZ RT-PCRreaction (PE Applied Biosystems) containing: approximately 25 ng oftotal RNA in DEPC treated water (Dnase/Rnase free); appropriate primers(approximately 800 nM ZC40541 (SEQ ID NO:87) and ZC40542 (SEQ ID NO:88);appropriate probe (approximately 100 nM ZC40544 (SEQ ID NO:89); 1×TaqMan EZ Buffer; 3 mM Manganese acetate; 300 μM each d-CTP, d-ATP, andd-GTP and 600 μM of d-UTP; rTth DNA Polymerase (0.1 U/μl); and AmpEraseUNG (0.01 U/μl). PCR thermal cycling conditions were as follows: aninitial UNG treatment step of one cycle at 50° C. for 2 minutes;followed by a reverse transcription (RT) step of one cycle at 60° C. for30 minutes; followed by a deactivation of UNG step of one cycle at 95°C. for 5 minutes; followed by 40 cycles of amplification at 94° C. for20 seconds and 60° C. for 1 minute.

Relative IL-20 RNA levels were determined by using the Standard CurveMethod as described by the manufacturer, PE Biosystems (User Bulletin#2: ABI Prism 7700 Sequence Detection System, Relative Quantitation ofGene Expression, Dec. 11, 1997). The hGUS measurements were used tonormalize IL-20 levels. Data are shown in Table 4 below. TABLE 4 SkinSample IL-20 Normal 2903 Uninvolved 7233 Involved 27,695

IL-20 mRNA was detectable in skin samples from normal patients or fromuninvolved areas. In contrast, there was upregulation for IL-20 messagein involved skin from psoriasis patients. IL-20RA and IL-20RB areexpressed in human normal and diseased skin. These data support a strongdisease association for IL-20 to human psoriasis.

Overexpression of IL-20 was shown in human psoriatic lesions, suggestingthat IL-20 is involved in human psoriasis. Moreover, as describedherein, over expression of IL-20 in transgenic mice showed epidermalthickening and immune cell involvement indicative of a psoriaticphenotype. Such in vivo data further suggests that IL-20 is involved inpsoriasis. As such, antagonists to IL-20 activity, such as theanti-human-IL-20, anti-human-IL-20RA and anti-human-IL-20RB monoclonalantibodies of the present invention, as well as soluble receptors andantibodies thereto, are useful therapeutically as antagonists to IL-20in the treatment of inflammatory diseases, such as psoriasis, as well asother indications as disclosed herein.

EXAMPLE 14 IL-20RA and IL-20RB mRNA are Up-Regulated in Psoriasis

In situ hybridization was used to determine whether IL-20 receptorexpression is altered in psoriasis. Skin samples from four psoriasispatients and three unaffected patients were assayed with probes specificfor the two-receptor subunit mRNAs. All four psoriatic skin samples hadhigh levels of IL-20RA and IL-20RB mRNA in keratinocytes whereas normalskin samples did not have detectable levels of either receptor subunitmRNA. Positive signals in psoriatic skin were also observed inmononuclear immune cells and in endothelial cells in a subset ofvessels. Therefore, both IL-20RA and IL-20RB are expressed inkeratinocytes, immune cells and endothelial cells, the major cell typesthought to interact in psoriasis.

EXAMPLE 15 IL-20 Binding Activates STAT3 in the HaCaT Keratinocyte CellLine

IL-20 binds cell lines transfected with both subunits of its receptor(i.e. IL-20RA and IL-20RB). However, these cell lines overexpress theIL-20 receptor relative to its normal level and their relevance to thephysiological role of IL-20 is unclear. The human HaCaT keratinocytecell line, which expresses endogenous IL-20RA and IL-20RB was used toexamine IL-20 signal transduction in a biologically relevant cell type.HaCaT cells were infected with recombinant adenovirus containing areporter construct to allow detection of intracellular signaling. Theconstruct consists of the firefly luciferase gene driven bypromoter/enhancer sequences comprised of the serum response element(SRE) and signal transducers and activators of transduction elements(STATs). This assay system detects productive ligand-receptorinteractions and indicates possible downstream signal transductioncomponents involved in receptor activation. Treatment with IL-20 aloneresulted in a dose-dependent increase in luciferase activity with a halfmaximal response occurring at approximately 2.3 nM. Subsequentluciferase reporter assays using adenovirus vectors containing only theSRE element or only the STAT elements produced detectable reporteractivation only through STATs.

To determine if other cytokines act in concert with IL-20, HaCaT cellswere treated with IL-20 alone or in combination with a single submaximaldose of EGF, IL-1β, or TNFα. In the presence of each of these threeproteins, IL-20 treatment resulted in a dose-dependent increase inluciferase activity. IL-20 in combination with IL-1β results in ahalf-maximal response at approximately 0.5 nM, about five-fold lowerthan with IL-20 alone. In addition, activation of the reporter gene isdetectable at 0.1 nM IL-20, a dose that is at least tenfold lower thanthe IL-20 dose required alone.

BHK cells transfected with IL-20RA, IL-20RB or both receptor subunitswere used to determine whether receptor pairing was required for IL-20stimulation of STAT-luciferase. As was the case with binding assays,only cells transfected with both receptor subunits responded to IL-20and did so with a half-maximal response of 5.7 pM. We note that theIL-20 concentration for the half-maximal response in BHK cells is400-fold lower than that for half-maximal response in HaCaT cells. It islikely that a lower concentration of IL-20 is needed for half-maximalresponse in BHK cells, as compared to HaCaT cells, due to higherreceptor levels in the BHK IL-20 receptor transfectants.

A nuclear translocation assay was used to identify STAT proteinsinvolved in IL-20 action. Both HaCaT cells, with endogenous IL-20receptors, and BHK cells transfected with IL-20RA and IL-20RB, weretreated with IL-20 protein and translocation of STAT3 and STAT1transcription factors from the cytoplasm to the nucleus was assayed byimmunofluorescence.

In unstimulated HaCaT cells, STAT3 staining was predominantly in thecytosol. Treatment of HaCaT cells with IL-20 resulted in a distinctaccumulation of STAT3 in the nucleus. Nuclear translocation of STAT3 inresponse to increasing concentrations of IL-20 occurred with ahalf-maximal IL-20 concentration of 7 nM. In contrast to STAT3translocation, HaCaT cells treated with IL-20 did not show anydetectable nuclear accumulation of STAT1.

BHK cells transfected with IL-20RA and UL-20RB were used to confirm thatthe IL-20 receptor was required for IL-20 stimulation of STAT3 nucleartranslocation. In BHK cells lacking the IL-20 receptor, STAT3 remainedcytosolic following treatment with IL-20. In contrast, in BHK cellstransfected with the IL-20 receptor, STAT3 translocated to the nucleusin response to IL-20. Again, STAT1 remained cytosolic regardless ofIL-20 treatment or IL-20 receptor expression. Thus, the IL-20 receptoris required for IL-20-mediated STAT3 activation.

EXAMPLE 16 IL-20 Transgenic Phenotype

Both human and mouse IL-20 were overexpressed in transgenic mice using avariety of promoters. The liver-specific mouse albumin promoter,directing expression of human IL-20, was used initially in an attempt toachieve circulating levels of protein. Subsequent studies were conductedusing the keratin 14 (K14) promoter, which primarily targets expressionto the epidermis and other stratified squamous epithelia; the mousemetallothionein-1 promoter, which gives a broad expression pattern; andthe E□LCK promoter, which drives expression in cells of the lymphoidlineage. Similar results were obtained in all four cases, possiblybecause these promoters all give rise to circulating levels of IL-20.

In all cases, transgenic pups expressing the IL-20 transgene weresmaller than non-transgenic littermates, had a shiny appearance withtight, wrinkled skin and died within the first few days after birth.Pups had milk in their stomachs indicating that they were able tosuckle. These mice had swollen extremities, tail, nostril and mouthregions and had difficulty moving. In addition, the mice were frail,lacked visible adipose tissue and had delayed ear and toe development.Low expression levels in liver (less than 100 mRNA molecules/cell) weresufficient for both the neonatal lethality and skin abnormalities.Transgenic mice without a visible phenotype either did not express thetransgene, did not express it at detectable levels, or were mosaic.

Histologic analysis of the skin of the IL-20 transgenic mice showed athickened epidermis, hyperkeratosis and a compact stratum corneumcompared to non-transgenic littermates. Serocellular crusts (scabs) wereobserved occasionally. Electron microscopic (EM) analysis of skin fromtransgenic mice showed intramitochondrial lipoid inclusions, mottledkeratohyaline granules, and relatively few tonofilaments similar to thatobserved in human psoriatic skin and in mouse skin disease models. Inaddition, many of the transgenic mice had apoptotic thymic lymphocytes.No other abnormalities were detected by histopathological analysis.These histological and EM results support and extend the observed grossskin alterations.

EXAMPLE 17 Anti-Human IL-20 Polyclonal Antibodies

Polyclonal antibodies were prepared by immunizing 2 female New Zealandwhite rabbits with the peptide, human IL-20X1-2(cgeeamkkyeqilshfeklepqaavvkalgeldillqw) (SEQ ID NO: 90) or the purifiedmature recombinant human polypeptide (SEQ ID NO:3) produced in BKHcells, human IL-20-BHK. The peptide was synthesized using an AppliedBiosystems Model 431A peptide synthesizer (Applied Biosystems, Inc.,Foster City, Calif.) according to manufacturer's instructions. Thesynthetic peptide human IL-20X1-2 was then conjugated to the carrierprotein maleimide-activated keyhole limpet hemocyanin (KLH) through theterminal cysteine residue (Pierce, Rockford, Ill.). The rabbits wereeach given an initial intraperitoneal (ip) injection of 200 μg of theconjugated synthetic peptide human IL-20X1-2 or the purified maturerecombinant polypeptide human IL-20-BHK in Complete Freund's Adjuvantfollowed by matched booster ip injections of 100 μg of the conjugatedpeptide or the mature polypeptide in Incomplete Freund's Adjuvant everythree weeks. Seven to ten days after the administration of the secondbooster injection (3 total injections), the animals were bled and theserum was collected. The animals were then boosted and bled every threeweeks.

The human IL-20X1-2 peptide-specific and human IL-20-BHKpolypeptide-specific rabbit sera were characterized by ELISA using 1μg/ml of the peptide human IL-20X1-2 or 500 ng/ml of the polypeptidehuman IL-20-BHK as specific antibody targets. The 4 rabbit serum sampleshad titer to their specific antibody targets at a dilution of 1:5E6(1:5,000,000).

The human IL-20X1-2 peptide-specific polyclonal antibodies were affinitypurified from appropriately pooled immune rabbit serum using anEPOXY-SEPHAROSE 6B peptide column (Pharmacia LKB) that was preparedusing 10 mg of the synthetic peptide human IL-20X1-2 per gram ofEPOXY-SEPHAROSE 6B. The human IL-20 polypeptide-specific polyclonalantibodies were affinity purified from appropriately pooled immunerabbit serum using a CNBr-SEPHAROSE 4B protein column (Pharmacia LKB)that was prepared using 10 mg of the purified mature recombinant humanpolypeptide produced in E.coli, human IL-20-E.coli per gram ofCNBr-SEPHAROSE. Following purification, the resulting polyclonalantibodies were dialyzed against 4 changes of 20 times the antibodyvolume of PBS over a time period of at least 8 hours.

Human IL-20X1-2 peptide and human IL-20 polypeptide-specific polyclonalantibodies were characterized by ELISA using 1 μg/ml of the syntheticpeptide human IL-20X1-2 or 500ng/ml of the purified recombinantpolypeptides, human IL-20-BHK, human IL-20-Bv, or human IL-20-E.coli asantibody targets. The human IL-20X1-2 peptide-specific polyclonalantibodies exhibited lower limits of detection (LLDs) of 100 pg/ml and500 pg/ml to its specific antigen human IL-20X1-2 and the polypeptidehuman IL-20-BHK, respectively. The human IL-20 polypeptide-specificpolyclonal antibodies exhibited LIDs of 100 pg/ml on the antigen targetshuman IL-20-BHK, human IL-20-Bv, and human IL-20-E.coli.

The human IL-20 polypeptide-specific affinity purified polyclonalantibodies were characterized further for their ability to block thecell-proliferative activity (“neutralization assay”) of purifiedrecombinant human IL-20 on BaF3/IL-20RA/IL-20RB cells. A 10× molarexcess of the human IL-20 polypeptide-specific polyclonal antibodies wassufficient to inhibit cell proliferation.

The human IL-20 polypeptide-specific affinity purified polyclonalantibodies were characterized for their utility in an ELISA for thequantitative determination of the recombinant mature polypeptides humanIL-20-BHK, human IL-20-Bv, or human IL-20-E.coli in mouse and humanserum samples. The resulting ELISA exhibited a lower limit of detectionof 1 ng/ml in 100% normal mouse serum and 5 ng/ml in 100% human serumfor all three forms of recombinant mature polypeptide human IL-20.

EXAMPLE 18 Construction of BaF3 Cells Expressing the KZ134 Plasmid andIL-20RA and IL-20RB Subunits

BaF3 cells expressing the KZ134 plasmid were constructed as describedbelow and designated BaF3/KZ134. These cells were used as a control andfurther transfected with full length IL-20RB (SEQ ID NO:20) as describedbelow. The BaF3/KZ134 cells expressing the IL-20RB were designated asBaF3/KZ134/IL-20RB. These cells were used as a control and furthertransfected with full length IL-20RA (SEQ ID NO:13) as described below.The BaF3/KZ134/IL-20RB cells expressing the IL-20RA were designated asBaF3/KZ134/IL-20RA/IL-20RB.

Construction of BaF3 Cells Expressing the KZ134 Plasmid

BaF3, an interleukin-3 (IL-3) dependent pre-lymphoid cell line derivedfrom murine bone marrow (Palacios and Steinmetz, Cell 41: 727-734, 1985;Mathey-Prevot et al., Mol. Cell Biol. 6: 4133-4135, 1986), wasmaintained in complete media (RPMI 1640 medium (JRH Bioscience Inc.,Lenexa, Kans.) supplemented with 10% heat-inactivated fetal bovineserum, 1 ng/ml murine IL-3 (mIL-3) (R & D, Minneapolis, Minn.), 2 mML-Glutamine (Gibco BRL), 1 mM Sodium Pyruvate (Gibco BRL), and PSNantibiotics (Gibco BRL)). The KZ134 plasmid is constructed withcomplementary oligonucleotides ZC12,749(gtaccttcccgtaaatccctccccttcccggaattacacccgcgtatttcccagaaaaggaactgtagatttctaggaattcaatccttggccacgcgtc) and ZC12,748(tcgagacgcgtggccaaggat tgaattcctagaaatctacagttccttttctgggaaatacgcgggtgtaattccgggaaggggagggatttacgggaag) that contain STAT transcription factorbinding elements from 4 genes. A modified c-fos Sis inducible element(m67SIE, or hSIE) (Sadowski, H. et al., Science 261:1739-1744, 1993),the p21 SIE1 form the p21 WAF1 gene (Chin, Y. et al., Science272:719-722, 1996), the mammary gland response element of the β-caseingene (Schmitt-Ney, M. et al., Mol. Cell. Biol. 11:3745-3755, 1991), anda STAT inducible element of the Fcg RI gene (Seidel, H. et al., Proc.Natl. Acad. Sci. 92:3041-3045, 1995). These oligonucleotides containAsp718-XhoI compatible ends and are ligated, using standard methods,into a recipient firefly luciferase reporter vector with a c-fospromoter (Poulsen, L. K. et al., J. Biol. Chem. 273:6229-6232, 1998)digested with the same enzymes and containing a neomycin selectablemarker. The KZ134 plasmid is used to stably transfect BaF3 cells, usingstandard transfection and selection methods (as describe below) with 500μg/ml neomycin, to make the BaF3/KZ134 cell line.

Construction of BaF3/KZ134 Cells Expressing the IL-20RB

The full-length cDNA sequence of IL-20RB (SEQ ID NO:20) was isolatedfrom a cDNA library, and then cloned into an expression vector pZP7P.Prior to electroporation, IL-20RB/pZP7P was prepared and purified usinga Qiagen Maxi Prep kit (Qiagen) as per manufacturer's instructions. Forelectroporation, BaF3/KZ134 cells were washed once in serum-free RPMImedia and then resuspended in serum-free RPMI media at a cell density of10⁷ cells/ml. One ml of resuspended BaF3/KZ134 cells was mixed with 30μg of the IL-20RB/pZP7P plasmid DNA and transferred to separatedisposable electroporation chambers (GIBCO BRL). Following a 15-minuteincubation at room temperature the cells were given two serial shocks(800 1Fad/300 V.; 1180 1Fad/300 V.) delivered by an electroporationapparatus (CELL-PORATOR™; GIBCO BRL). After a 5-minute recovery time,the electroporated cells were transferred to 50 ml of complete media andplaced in an incubator for 15-24 hours at 37° C. and 5% CO₂. The cellswere then spun down and resuspended in 50 ml of complete mediacontaining 500 μg/ml neomycin and 2 μg/ml puromycin in a T-162 flask toisolate the puromycin-resistant pool. Pools of the transfectedBaF3/KZ134 cells, hereinafter called BaF3/KZ134/IL-20RB, were assayedfor signaling capability as described below. Moreover these cells werefurther transfected with IL-20RA as described below. BaF3/KZ134/IL-20RAcells were also made as described above.

Construction of BaF3/KZ134/IL-20RB Cells Expressing the IL-20RA

BaF3/KZ134/IL-20RB cells expressing the full-length IL-20RA (SEQ IDNO:14) were constructed as per above, using 30 μg of a IL-20RA/pZP7Zexpression vector. Following recovery, transfectants were selected using500 μg/ml neomycin, 2 μg/ml puromycin, and 200 μg/ml zeocin to isolatethe zeocin-resistant pool. Pools of the transfected BaF3/KZ134/IL-20RBcells were diluted and plated out using standard techniques. Individualclones were screened by luciferase assay described in U.S. Ser. No.09/745,792, Example 13, which is incorporated herein in its entirety,using purified recombinant human IL-20x1-Bv as an inducer. Clones withthe highest luciferase response (via STAT luciferase) and the lowestbackground were selected. Hereinafter the transfected cell line iscalled BaF3/KZ134/1L-20RA/IL-20RB.

Similarly, a BHK cell line was also constructed using the methoddescribed herein, and can be used in luciferase assay described above.The cell line is called BHK/KZ134/IL-20RA/IL-20RB.

EXAMPLE 19 Screening for IL-20 Activity Using BaF3/KZ134/IL-20RA/IL-20RBProliferation Assay

Purified recombinant human IL-20x1-Bv was used to test for the presenceof proliferative activity as described below. BaF3/KZ134/IL-20RA/IL-20RBcells were spun down and washed in BaF3 assay media (RPMI 1640 medium(JRH Bioscience Inc., Lenexa, Kans.) supplemented with 10%heat-inactivated fetal bovine serum, 2 mM L-Glutamine (Gibco BRL), 1 mMSodium Pyruvate (Gibco BRL), and PSN antibiotics (Gibco BRL); withoutmIL-3 and neomycin, puromycin, and zeocin selection (hereinafterreferred to as BaF3 assay media)). The cells were spun down and washed 3times in BaF3 assay media to ensure the removal of the mIL-3.BaF3/KZ134/IL-20RA/IL-20RB cells were then counted and plated out in96-well format at 5000 cells per well in 100 μl of BaF3 assay media.Serial dilutions of IL-20 ranging from 1 pM to 1 nM were added to theBaF3/KZ134/IL-20RA/IL-20RB cells in 100 μl of BaF3 assay media. Totalassay volume is 200 μl per well. The assay plates were incubated at 37°C. and 5% CO₂ for 72 hours at which time Alamar Blue (Accumed, Chicago,Ill.) was added at 20 μl per well. Plates were again incubated at 37° C.and 5% CO₂ for 24 hours. Alamar Blue gives a fluourometric readout basedon the number of live cells, and is thus a direct measurement of cellproliferation in comparison to a negative control (BaF3 assay mediaalone). Plates were read on the fmax™ plate reader (Molecular Devices,Sunnyvale, Calif.) using the Softmax™ Pro program at wavelengths 544 nmexcitation and 590 nm emission. Results confirmed the dose dependentproliferative response of the BaF3/KZ134/IL-20RA/IL-20RB cells to IL-20.The response, as measured, was approximately 30-fold over background atthe high end of 1 nM down to a 2-fold over background at the low end of1 pM. The BaF3/KZ134 parental cells, the BaF3/KZ134/IL-20RA only cells,and the BaF3/KZ134/IL-20RB only cells did not proliferate in response toIL-20, showing that IL-20 is specific for the IL-20RA/IL-20RBheterodimeric receptor. The BaF3/KZ134/IL-20RA/IL-20RB alamar blueproliferation assay can also be used to look for antagonists to IL-20,by measuring the abstinence of the cell-proliferative response of IL-20when run in combination with antagonists (“neutralization assay”).

EXAMPLE 20 Anti-Human IL-20 Monoclonal Antibodies

Rat monoclonal antibodies were prepared by immunizing 4 femaleSprague-Dawley Rats (Charles River Laboratories, Wilmington, Mass.),with the purified mature recombinant polypeptide (SEQ ID NO:3) producedin Baculovirus, human IL-20-Bv. The rats were each given an initialintraperitoneal (IP) injection of 25 μg of the purified recombinantpolypeptide in Complete Freund's Adjuvant (Pierce, Rockford, Ill.)followed by booster IP injections of 10 μg of the purified recombinantpolypeptide in Incomplete Freund's Adjuvant every two weeks. Seven daysafter the administration of the second booster injection, the animalswere bled and serum was collected.

The human IL-20-specific rat serum samples were characterized by ELISAusing 1 ug/ml of the purified mature recombinant polypeptide humanIL-20-Bv as the specific antibody target. Three rat serum samples hadtiter to the specific antibody target at a dilution of 1:1E6. One ratserum sample had titer to the specific antibody target at a dilution of1:1E4.

Splenocytes were harvested from a single high-titer rat and fused toSP2/0 (mouse) myeloma cells using PEG 1500 in a single fusion procedure(4:1 fusion ratio, splenocytes to myeloma cells, “Antibodies: ALaboratory Manual, E. Harlow and D.Lane, Cold Spring Harbor Press).Following 9 days growth post-fusion, specific antibody-producinghybridoma pools were identified by radioimmunoprecipitation (RIP) usingthe Iodine-125 labeled recombinant polypeptide human IL-20-Bv as thespecific antibody target and by ELISA using 500 ng/ml of the recombinantpolypeptide human IL-20-Bv as the specific antibody target. Hybridomapools positive in either assay protocol were analyzed further for theirability to block the cell-proliferative activity (“neutralizationassay”) of purified recombinant polypeptide human IL-20-Bv onBaf3/IL-20RA/IL-20RB cells.

Hybridoma pools yielding positive results by RIP only or RIP and the“neutralization assay” were cloned at least two times by limitingdilution.

Monoclonal antibodies purified from tissue culture media werecharacterized for their ability to block the cell-proliferative activity(“neutralization assay”) of purified recombinant human IL-20 on Baf3cells expressing both receptor sequences human IL-20RA and humanIL-20RB. Three “neutralizing” monoclonal antibodies were identified inthis manner.

Hybridomas expressing the neutralizing monoclonal antibodies to humanIL-20 described above were deposited with the American Type TissueCulture Collection (ATCC; Manassas Va.) patent depository as originaldeposits under the Budapest Treaty and were given the following ATCCAccession No.s: 262.4.1.2.2.1 (ATCC [PTA-5350]); 262.5.1.6.4.4 (ATCC[PTA-5351]); 262.7.1.3.2.4 (ATCC [PTA-5352]).

EXAMPLE 21 Screening for IL-20 Antagonist Activity usingBaF3/KZ134/IL-20RA/IL-20RB Proliferation Assay

In order to determine if rat anti-human IL-20 monoclonal antibodies arecapable of antagonizing purified recombinant human IL-20x1-Bv, hybridomapools positive in the RIP assay were analyzed further for their abilityto block the cell-proliferative activity (“neutralization assay”) ofIL-20 on BaF3/KZ134/IL-20RA/IL-20RB cells (Example 18).

Monoclonal antibodies purified from tissue culture media werecharacterized for their ability to block the cell-proliferative activity(“neutralization assay”) of purified recombinant human IL-20x1-E.coli onBaF3/KZ134/IL-20RA/IL-20RB cells. Results are given as EC₅₀ and EC₁₀₀values in Table 5 below. TABLE 5 Rat anti-human IL-20 MonoclonalAntibody EC₅₀ EC₁₀₀ 262.4.1.2.2.1 0.34 nM   1 nM 262.5.1.6.4.4 2.54 nM 10 nM 262.7.1.3.2.4 0.19 nM 0.3 nMEC₅₀ (effective concentration at 50 percent)EC₁₀₀ (effective concentration at 100 percent)

EXAMPLE 22 Determining the Binding Affinity (K_(d)) for the RatAnti-Human IL-20 Monoclonal Antibodies to IL-20

Preparation of ¹²⁵I-IL-20. Radiolabeled purified recombinant humanIL-20x1-E.coli was prepared with Iodo-Beads® Iodination Reagent (Pierce)according to manufacturer's instructions. Twenty μg of IL-20 wasradiolabeled to specific activities of 45,000 to 137,000 counts perminute per nano gram with 95 to 100 percent of the radioactivityprecipitating with 10 percent TCA. Bioactivity of each preparation of¹²⁵I-IL-20 was measured using BHK/KZ134/IL-20RA/IL-20RB cells forluciferase response (via STAT luciferase) (U.S. Ser. No. 09/745,792).There was no significant differences in bioactivities of the 125,labeled IL-20 and unlabeled IL-20.

Binding of ¹²⁵I-IL-20 to the rat anti-human IL-20 monoclonal antibodies(Example ?). BreakApart Module 96 well micro titer plates (NUNC BrandProducts, Roskilde Denmark) were coated overnight at 4° C. with the ratanti-human IL-20 monoclonal antibodies at a 2 nM concentration in 100 μlper well of ELISA A (0.1M Sodium Carbonate Buffer, pH 9.6). Two nMcoating concentration of antibody was optimized for assay conditions.Plates were washed two times using automated plate washer (SLT 96PW),300 μl per well with ELISA C (0.05% Tween 20/1× Phosphate BufferedSaline). Plates were blocked for 5 minutes with 200 μl per wellSuperBlock® Blocking Buffer (Pierce), repeated. Plates were washed asdescribed above using automated plate washer. Binding was performed at37° C. with shaking for 3.0 hours using serial dilutions of ¹²⁵I-IL-20ranging from 3.3 μM to 2 nM concentration in 100 μl per well of ELISA B(1% Bovine Serum Albumin Fraction IV/0.05% Tween 20/1× PhosphateBuffered Saline). Specific binding was determined in the presence(nonspecific binding) and absence (total binding) of unlabeled IL-20 at2 μM concentration. Three hour time point, serial dilutions of¹²⁵I-IL-20, and 2 μM unlabeled IL-20 concentration, were all optimizedfor assay conditions. Binding reactions were terminated by removal ofthe binding medium and washing of the plates manually four times with200 μl per well of ELISA C. Plates were then broken apart and individualwells read on a gamma counter (Packard, Meriden, Conn.) for counts perminute incorporated.

For all three rat anti-human IL-20 monoclonal antibodies, specificbinding curves were generated using GraphPad Prism® (GraphPad SoftwareInc., San Diego, Calif.). The specific binding data was directly fittedusing non-linear regression to evaluate the K_(d) of ¹²⁵I-IL-20 to eachof the rat anti-hu-IL-20 monoclonal antibodies, as shown in Table 6below. TABLE 6 Rat anti-human IL-20 Monoclonal Antibody K_(d) of¹²⁵I-IL-20 262.4.1.2.2.1 0.074 nM 262.5.1.6.4.4 0.274 nM 262.7.1.3.2.40.128 nM

EXAMPLE 23 Human IL-20RA Monoclonal Antibodies

Mouse monoclonal antibodies were prepared by immunizing 5 female Balb Cmice (Charles River Laboratories, Wilmington, Mass.), with the purifiedrecombinant protein, IL-20RA-BHK. The mice were each given an initialintraperitoneal (IP) injection of 20 μg of the purified recombinantprotein in Complete Freund's Adjuvant (Pierce, Rockford, Ill.) followedby booster IP injections of 10 μg of the purified recombinant protein inIncomplete Freund's Adjuvant every two weeks. Five days after theadministration of the second booster injection, the animals were bledand serum was collected.

The IL-20RA-specific mouse sera samples were characterized by ELISAusing 500 ng/ml of the purified recombinant protein IL-20RA/RBReceptor-Ig Fusion heterodimer as specific antibody target. All 5 mouseserum samples had titer by ELISA to the specific antibody target at adilution of 1:1E6.

Splenocytes and lymph nodes were harvested from two high-titer mice andfused to the P3-X63-Ag8.653 mouse myeloma cell line in a single fusionprocedure (2.3:1 fusion ratio, Hope Heart Institute Contract AntibodyDevelopment, Journal of Immunological Methods 81, 223-228). Following 9or 10 days growth post-fusion, specific antibody-producing hybridomapools were identified by ELISA using 1 μg/ml of the purified recombinantprotein IL-20RA Receptor-Ig Fusion homodimer as the specific antibodytarget. IL-20RA-specific hybridoma pools were analyzed further by ELISAusing 1 μg/ml of the purified recombinant protein IL-20RA/IL-20RBReceptor-Ig Fusion heterodimer and by FACS for their ability to bind toBaf3/KZ55/IL-20RA/IL-20RB cells.

Hybridoma pools yielding positive results by the ELISA and the FACSassay were analyzed for their ability to block the cell-proliferativeactivity (‘neutralization assay’) of the purified recombinant proteinhuman IL-20-Bv on Baf3/KZ55/IL-20RA/IL-20RB cells. One ‘neutralizing’hybridoma pool was identified in this manner.

Hybridoma pools yielding positive results by the ELISA and FACS assayswere cloned at least two times by limiting dilution.

Monoclonal antibodies purified from tissue culture media werecharacterized by FACS for their ability to bind to human monocytic celllines THP-1 ATCC# TIB-202, HL-60, and U937 and to human monocytes inblood. One positive-binding monoclonal antibody was identified andisolated.

EXAMPLE 24 Human IL-20RB Polyclonal Antibodies

Anti IL-20RB polyclonal antibodies were prepared by immunizing 2 femaleNew Zealand white rabbits with the purified mature recombinant humanIL-20RB receptor-Ig fusion homodimer. The rabbits were each given aninitial intraperitoneal (ip) injection of 200 μg of purified protein inComplete Freund's Adjuvant followed by booster IP injections of 100 μgpeptide in Incomplete Freund's Adjuvant every three weeks. Seven to tendays after the administration of the second booster injection (3 totalinjections), the animals were bled and the serum was collected. Theanimals were then boosted and bled every three weeks.

The human IL-20RA-specific polyclonal antibodies were affinity purifiedfrom the immune rabbit serum using a CNBr-SEPHAROSE 4B protein column(Pharmacia LKB) that was prepared using 10 mg of the specific antigenthrombin-cleaved and purified recombinant human IL-20RB receptor-Igfusion homodimer per gram of CNBr-SEPHAROSE. Following purification, thepolyclonal antibodies were dialyzed with 4 changes of 20× the antibodyvolume of PBS over a period of at least 8 hours. Human IL-20RB-specificantibodies were characterized by ELISA using 500 ng/ml of the purifiedrecombinant protein human IL-20RB receptor-Ig fusion heterotetramer asthe antibody target. The lower limit of detection (LID) of the rabbitanti-human IL-20RB affinity purified antibody was 500 pg/ml on itsspecific antigen.

The human IL-20-specific polyclonal antibodies were characterizedfurther for their ability to block the cell-proliferative activity(“neutralization assay”) of purified recombinant human IL-20-BHK onBaF3/IL-20RA/IL-20RB cells (as described herein). A 10× molar excess ofthe human IL-20-specific polyclonal antibodies was sufficient to inhibitcell proliferation. The human IL-20RB-specific polyclonal antibodieswere also characterized further for their utility in an ELISA for thequantative determination of purified recombinant human IL-20RA/IL-20RBreceptor-Ig fusion heterotetramer in SCID mouse serum samples. Theresulting ELISA exhibited a lower limit of detection of 20.6 ng/ml in100% SCID mouse serum.

EXAMPLE 25 Human IL-20RB Monoclonal Antibodies

Rat monoclonal antibodies were prepared by immunizing 4 femaleSprague-Dawley Rats (Charles River Laboratories, Wilmington, Mass.),with the thrombin-cleaved and purified recombinant protein, humanIL-20RB Receptor-Ig Fusion homodimer. The rats were each given aninitial intraperitoneal (IP) injection of 100 μg of the purifiedrecombinant protein in Complete Freund's Adjuvant (Pierce, Rockford,Ill.) followed by booster EP injections of 50 μg of the purifiedrecombinant protein in Incomplete Freund's Adjuvant every two weeks.Seven days after the administration of the second booster injection, theanimals were bled and serum was collected.

The human IL-20RB-specific rat sera samples were characterized by ELISAand FACS using 500 ng/ml of the cleaved purified recombinant proteinhuman IL-20RB Receptor-Ig Fusion homodimer or Baf3/KZ55/IL-20RA/IL-20RBcells as specific antibody targets. All 4 rat serum samples had titer byELISA to the specific antibody target at a dilution of 1:1E6. Two ratserum samples had titer by FACS to the Baf3/KZ55/IL-20RA/IL-20RB cellsat a dilution of 1:1E3.

Splenocytes were harvested from a single high-titer rat and fused toSP2/0 (mouse) myeloma cells using PEG 1500 in a single fusion procedure(4:1 fusion ratio, splenocytes to myeloma cells, ‘Antibodies: ALaboratory Manual’, E. Harlow and D.Lane, Cold Spring Harbor Press).Following 10 days growth post-fusion, specific antibody-producinghybridoma pools were identified by ELISA using 1 μg/ml of the purifiedrecombinant protein human IL20-RA/RB Receptor-Ig Fusion heterodimer asthe specific antibody target and 1 μg/ml of an unrelated purifiedrecombinant human Receptor-Ig Fusion protein as the non-specific target.IL-20RB-specific hybridoma pools were analyzed further by FACS for theirability to bind to Baf3/KZ55/IL-20RA/IL-20RB cells.

Hybridoma pools yielding positive results by either the ELISA or theFACS assay were analyzed for their ability to block thecell-proliferative activity (‘neutralization assay’) of the purifiedrecombinant protein human IL-20-Bv on Baf3/KZ55/WL-20RA/IL-20RB cells.Seven ‘neutralizing’ hybridoma pools were identified in this manner.

Hybridoma pools yielding positive results by ELISA and FACS assays werecloned at least three times by limiting dilution.

Monoclonal antibodies purified from tissue culture media werecharacterized by FACS for their ability to bind to the human acutemonocytic leukemia cell line THP-1 ATCC# TEB-202. Two positive-bindingmonoclonal antibodies were identified.

EXAMPLE 26 IL-20 Antagonists in CD4⁺CD45RB^(hi) (CD25⁻) Colitis andPsoriasis Model

Transfer of CD4+ CD45RB^(hi) or CD4+CD25− T cells into syngeneic SCIDmice results in colitis in the mice. Co-transfer of regulatory T cells(CD4+CD25+ or CD4+CD45RB^(lo)) inhibits this colitis. After transfer ofCD4+CD25− T cells into mice, if mice are additionally injected withstaphylococcal enterotoxin B (SEB), mice not only develop colitis, butalso psoriasis. Antibodies against IL-20, IL-20RA and/or IL-20RB, orsoluble IL-20RA, IL-20RB or IL-20RA/IL-20RB receptors are administeredfrom days 0-21 after cell transfer and symptoms for colitis andpsoriasis are monitored. Inhibition of psoriatic score or colitis(histology) indicates that antibodies against IL-20, IL-20RA and/orIL-20RB, or soluble IL-20RA, IL-20RB or IL-20RA/IL-20RB receptors caninhibit these autoimmune diseases.

Study Design

Spleens and inguinal lymph nodes are isolated from B10.D2 mice. Singlecell suspensions are formed and counted. Using the Miltenyi Bead system,CD25+ cells are sorted out by positive selection. Cells are stained withCD25-PE (BD Pharmingen) at 1:100 dilution and incubated for 15 minutes.Excess antibody is washed out and the cells are incubated with 10 ulanti-PE beads/10⁶ cells for 20 minutes. The cells are washed with PBSand passed over an LS column (Miltenyi Biotech). Cells that pass throughthe column (CD25−) are retained for further analysis. A CD4 enrichmentcocktail (Stem Cell technologies) is added (1:100) to these CD25− cellsand incubated for 15 minutes. Cells are washed with PBS. A 1:10 dilutionof anti-biotin tetramer is added to the cells for 15 minutes followed bya magnetic colloid (60 ul/10⁶ cells) for 15 minutes (all from Stem CellTechnologies). Cells are passed through a negative selection column(0.5″, Stem cell Technologies). Cells that pass through are theCD4+CD25− cells. Purity is analyzed using flow cytometry. 0.4×10⁶ cellsare injected i.v into naive CB-17 SCID mice in a total volume of 200 μl.Mice are injected i.p with 10 μg SEB the following day (dl). Symptomsfor psoriasis and colitis are followed from 2-5 weeks. Mice are scoredfor psoriasis disease under the following criteria. 0-no lesions, 1—mildlesions on the neck, 2—severe lesions on the neck and back (trunk)3—very severe lesions on the neck, back and the belly of mice. Earthickening is also measured as a measure of disease severity. Groups ofmice are injected i.p. with PBS, 100 μg control antibody or 10-100 μgantibodies against IL-20, IL-20RA and/or IL-20RB, or soluble IL-20RA,IL-20RB or IL-20RA/IL-20RB from days 1-30 under different dosing regimen(3×/week or 2×/week).

Results and Conclusion

Inhibiton of psoriatic and colitis symptoms in antibody treated miceindicates that inhibition of IL-20 function can inhibit autoimmunesymptoms in this model for psoriasis and colitis.

EXAMPLE 27 Screening for IL-20 Antagonist Activity in an Alamar BlueProliferation Assay

The factor-dependent pre-B cell line BaF3 was co-transfected withIL-20RA and IL-20RB and treated with IL-20 at various concentrations.Proliferation was assessed busing and alamar blue assay. IL-20stimulated proliferation in a dose-dependent manner at concentrationsexpected for a cytokine, demonstrating that IL-20 binds and activatesthe heterodimeric IL-20RA/IL-20RB receptor at concentrations expectedfor a cytokine. The negative controls containing untransfected BaF3 didnot proliferate.

In order to determine if anti-IL-20RA antibodies are capable ofantagonizing IL-20 activity, the assay described above is performedusing either anti-il-20 antibodies, anti-IL-20RA antibodies oranti-IL-20RB antibodies as an antagonist to IL-20 activity. When IL-20is combined with such antagonist, the response to IL-20 at allconcentrations is brought down to background levels. That the presenceof an antagonist that ablates or reduces the proliferative effects ofIL-20 demonstrates that it is an antagonist of the IL-20 ligand. Thisassay can be used to test other antagonists of IL-20 activity describedherein, such as soluble IL-20RA, IL-20RB or IL-20RA/IL-20RB receptor.

EXAMPLE 28 Anti-IL-20 mAbs, Anti-IL-20RA mAbs or Anti-IL-20RB mAbsInhibit Disease Severity in a Mouse CIA Model

The collagen-induced arthritis (CIA) model is a mouse model forrheumatoid arthritis that reflects to large extent the disease seen inhumans. (Moore, Methods Mol. Biol. 225:175-179, 2003: Waksman, Scand. J.Immunol., 56:12-34, 2002). Mice are immunized with 2 doses of collagenemulsified in CFA at the base of the tail. This results in swelling ofthe paws that increases over a period of time and can be both visuallyscored and measured using calipers. Furthermore, serum anti-collagenantibodies correlates well with severity of disease. Based on datashowing IL-20 and IL-22 induce inflammation, anti-IL-20, anti-IL-20RAand anti-IL-20RB mAbs are administered separately or in any combinationthereof (i.e. anti-IL-20 mAbs in combination with anti-IL-20RA and/oranti-IL-20RB mAbs; or anti-IL-20RA mAbs in combination withanti-IL-20RB) to groups of collagen-immunized mice, and effects ondisease scores are evaluated. A decrease in paw scores and paw thicknessafter administration of any of these mAbs suggests IL-20 promotesongoing immune response in a model for autoimmunity and blocking IL-20'sfunction may inhibit autoimmune disorders. Inhibition of serum TNFa andanti-collagen antibodies also suggests that blocking IL-20, IL-20RAand/or IL-20RB may be beneficial in autoimmune disease.

Thus, to determine if anti-IL-20, anti-IL-20RA and anti-IL-20RB mAbshave an effect on autoimmunity, they are tested in a mouse model forrheumatoid arthritis—collagen-induced arthritis (CIA). Specifically,DBA1J mice are given collagen injections to induce rheumatoid-likearthritis. The inoculation on Day 0 is a subcutaneous injection of ahomogenate consisting of Complete Freund's Adjuvant (CFA) and Type IIcollagen (50-100 μl, prepared as 2 mg/ml of collagen). The injection isgiven near the base of the tail. On Day 21, a second inoculation isadministered, the only difference being that the homogenate is preparedusing Incomplete Freund's Adjuvant (IFA), instead of the CFA. Paw scoresand thickness are measured daily. Groups of mice receive PBS, 20-200 ugcontrol isotype matched monoclonal antibody or 20-200 ug anti-IL-20,anti-IL-20RA and anti-IL-20RB mAb i.p 2× or 3×/week for 1-4 weeksstarting at second collagen injection. Mice are monitored daily till day30. Mice are sacrificed on day 30, serum taken for anti-collagenantibody analysis and serum cytokine analysis (TNF-alpha).

Inhibition of paw scores, paw thickness, serum TNF-alpha and serumanti-collagen antibodies by administration of anti-IL-20, anti-IL-20RAand anti-IL-20RB mAbs suggests that blocking IL-20, IL-20RA and/orIL-20RB can inhibit an ongoing immune response in a model forautoimmunity and may inhibit autoimmune disorders.

EXAMPLE 29 IL-20 is Up-Regulated in Human Atopic Dermatitis Skin Samples

RNA Samples

Normal skin samples as well as skin from atopic dermatitis patients wereobtained. RNA was isolated from human skin samples using conventionalmethods. The integrity and quality of RNA samples was tested on theAgilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn Germany).

Primers and Probes for Quantitative RT-PCR

Real-time quantitative RT-PCR using the ABI PRISM 7700 SequenceDetection System (PE Applied Biosystems, Inc., Foster City, Calif.) hasbeen previously described (See, Heid, C. A. et al., Genome Research6:986-994, 1996; Gibson, U. E. M. et al., Genome Research 6:995-1001,1996; Sundaresan, S. et al., Endocrinology 139:4756-4764, 1 998. Thismethod incorporates use of a gene specific probe containing bothreporter and quencher fluorescent dyes. When the probe is intact thereporter dye emission is negated due to the close proximity of thequencher dye. During PCR extension using additional gene-specificforward and reverse primers, the probe is cleaved by the 5′ to 3′nucleolytic activity of the rTth DNA Polymerase which releases thereporter dye from the probe resulting in an increase in fluorescentemission.

The primers and probes used for real-time quantitative RT-PCR analysesof IL-20 expression were designed using the primer design softwarePrimer Express™ (PE Applied Biosystems, Foster City, Calif.). Theforward primer, ZC40541 (SEQ ID NO:25) and the reverse primer, ZC 40542(SEQ ID NO:26) were used in a PCR reaction (below) at a 800 nMconcentration to synthesize a 71 bp product. The corresponding IL-20TaqMan® probe, ZC 40544 (SEQ ID NO:27) was synthesized and labeled by PEApplied Biosystems. The IL-20 probe was labeled at the 5′ end with areporter fluorescent dye (6-carboxy-fluorescein) (FAM) (PE AppliedBiosystems) and at the 3′ end with a quencher fluorescent dye(6-carboxy-tetramethyl-rhodamine) (TAMRA) (PE Applied Biosystems).

Real-Time Quantitative RT-PCR

Relative levels of IL-20 mRNA were determined by analyzing total RNAsamples using the TaqMan EZ RT-PCR Core Reagents Kit (PE AppliedBiosystems). Runoff IL-20 transcript was made to generate a standardcurve used for quantitation. The curve consisted of 10-fold serialdilutions ranging from 1e8 to 1e3 total copies of whole message forIL-20 with each standard curve point analyzed in triplicate. The totalRNA samples from skin were also analyzed in triplicate for human IL-20transcript levels and for levels of hGUS as an endogenous control. In atotal volume of 25 μl, each RNA sample was subjected to TaqMan EZ RT-PCRreaction (PE Applied Biosystems) containing: approximately 25 ng oftotal RNA in DEPC treated water (Dnase/Rnase free); appropriate primers(approximately 800 nM ZC40541 (SEQ ID NO:25) and ZC40542 (SEQ ID NO:26);appropriate probe (approximately 100 nM ZC40544 (SEQ ID NO:27); 1×TaqMan EZ Buffer; 3 mM Manganese acetate; 300 μM each d-CTP, d-ATP, andd-GTP and 600 μM of d-UTP; rTth DNA Polymerase (0.1 U/μl); and AmpEraseUNG (0.01 U/μl). PCR thermal cycling conditions were as follows: aninitial UNG treatment step of one cycle at 50° C. for 2 minutes;followed by a reverse transcription (RT) step of one cycle at 60° C. for30 minutes; followed by a deactivation of UNG step of one cycle at 95°C. for 5 minutes; followed by 40 cycles of amplification at 94° C. for20 seconds and 60° C. for 1 minute.

Relative IL-20 RNA levels were determined by using the Standard CurveMethod as described by the manufacturer, PE Biosystems (User Bulletin#2: ABI Prism 7700 Sequence Detection System, Relative Quantitation ofGene Expression, Dec. 11, 1997). The hGUS measurements were used tonormalize IL-20 levels.

IL-20 mRNA was detectable at a low level (796 copies) in skin samples.In contrast, there was upregulation for IL-20 message in skins fromatopic dermatitis patients (8598 copies). The receptor subunits forIL-20, including IL-20RA), IL-20RA, and IL-20RB are expressed in humannormal and diseased skin. These data support a strong diseaseassociation for IL-20 to human atopic dermatitis.

Overexpression of IL-20 was shown in human atopic dermatitis skins,suggesting that IL-20 is involved in human atopic dermatitis. Moreover,as described herein, over expression of IL-20 in transgenic mice showedepidermal thickening and immune cell involvement indicative of an atopicdermatitis phenotype. Such in vivo data further suggests that IL-20 isinvolved in atopic dermatitis. As such, antagonists to IL-20 activity,such as the anti-human-IL-20RA monoclonal antibodies of the presentinvention, as well as soluble receptors and antibodies thereto, areuseful therapeutically as antagonists to IL-20 in the treatment ofinflammatory diseases, such as atopic dermatitis, as well as otherindications as disclosed herein.

EXAMPLE 30 Pharmacokinetics of an Anti-Human IL-20 Monoclonal Antibody

The test monoclonal antibody, anti-human IL-20 mAb, (clone#262.7.1.3.2.4; kd=0.133 nM) was provided in 3×3 mL aliquots at aconcentration of 1.08 mg/mL (determined by UV Absorbance at 280 nM) andwas stored at −80° C. until use. The vehicle was 1×PBS (50 mM NaPO4, 109mM NaCl), pH 7.3. The mAb was thawed at room temperature before use andaliquots 1 and 2 were used as provided for the 100/g IV and SC dosinggroups, respectively. Half of aliquot 3 was diluted 1:2 in 1×PBS for the50 μg SC dose group and the second half of aliquot 3 was diluted 1:10 in1×PBS for the 10 μg SC dose group. Female SCID mice (n=96), werereceived from Charles River Labs. Animals were checked for health onarrival and group-housed (3 animals per cage). The mice were 12 weeksold with an average body weight of 22 g at the beginning of the study.

Dosing Protocol

Female SCID mice (n=24/dose group) were randomly placed into four dosinggroups (Table 7). Group 1 was administered the anti-human IL-20 mAb viaIV injection of approximately 93 μL in a tail vein and Groups 2, 3, and4 were administered the mAb via SC injection of approximately 93 μL inthe scruff of the neck.

Sample Collection

Prior to blood collection, mice were fully anesthetized with halothaneor isofluorane. Blood samples were collected via cardiac stick for alltimepoints except the 168 hr timepoint (collected via eye bleed and thesame animals were bled again at the 504 hr timepoint via cardiac stick).Blood was collected into serum separator tubes and allowed to clot for15 minutes. Samples were subsequently centrifuged for 3 minutes at14,000 rpm. Following centrifugation, aliquots of 125-150 uL weredispensed into labeled eppendorf tubes and immediately stored at −80° C.until analysis (Table 7). TABLE 7 Dose Group # (ROA) Animals PKTimepoints 1 100 μg (IV) 3 mice/timepoint* 0.25, 1, 4, 8, 24, 72, 168,336 and 504 hr 2 100 μg (SC) 3 mice/timepoint* 0.25, 1, 4, 8, 24, 72,168, 336 and 504 hr 3  50 μg (SC) 3 mice/timepoint* 0.25, 1, 4, 8, 24,72, 168, 336 and 504 hr 4  10 μg (SC) 3 mice/timepoint* 0.25, 1, 4, 8,24, 72, 168, 336 and 504 hr*The same animals were used for the 168 and 504 hr timepoints.Quantification of Serum Anti-huIL-20 mAb Concentrations by ELISA

An Enzyme Linked Immunosorbant Assay (ELISA) was developed and qualifiedto analyze mouse serum samples from animals dosed with rat anti-IL-20mAb 267.7.1.3.2.4 during pharmacokinetic studies. This assay wasdesigned to take advantage of a commercially available secondaryantibody and calorimetric detection using TMB. The dilutions used forthe standard curve were modified to improve the definition of the linearportion of the standard curve. A standard curve in the range of 100ng/mL to 0.231 ng/mL with 2-fold dilutions allowed for quantitation ofthe mouse serum samples. QC samples were diluted to 1:100, 1:1000 and1:10000 in 10% SCID mouse serum and back calculated from the standardcurve.

Pharmacokinetic Analysis

Serum concentration versus time data were downloaded into WinNonlinProfessional 4.0 software (Pharsight, Inc.; Cary, N.C.) forpharmacokinetic analysis. Noncomparmental analysis was used to determinepharmacokinetic parameters based on the mean data at each time point.

Results

Mean serum anti-human IL-20 mAb concentrations following administrationof 100 μg IV and 100, 50, and 10 μg SC are shown in Table 8. TABLE 8Time 100 μg IV 10 μg SC 50 μg SC 100 μg SC (hr) Conc (μg/mL) Conc(μg/mL) Conc (μg/mL) Conc (μg/mL) 0.25  196 (12) LTR 0.101 (0.065) 0.481(0.485) 1  154 (18) 0.356 (0.146)  1.61 (0.52)  3.48 (1.72) 4  118 (20) 2.42 (0.53)  10.4 (3.4)  19.7 (4.7) 8  112 (20)  3.41 (0.30)  18.9(3.6)  40.2 (6.4) 24  103 (13)  4.95 (0.05)  26.3 (0.7)  50.1 (6.2) 72 101 (16)  4.27 (0.79)  21.0 (3.4)  43.4 (2.7) 168 45.6 (15.4)  2.92(0.53)  19.6 (2.7)  37.6 (3.4) 336 36.4 (16.6)  3.60 (0.31)  23.5 (3.5) 34.4 (5.8) 504 28.8 (3.8)  2.74 (0.39)  20.5 (3.6)  25.7 (2.1)LTR: less than reportable

Following IV administration, the mAb concentration versus time profiledemonstrated a biexponential decline. Following SC administration, themAb appeared to have a slow absorption phase, with absorptionrate-limited elimination. The serum pharmacokinetic parameters based onthe mean data at each time point are shown in Table 9. TABLE 9 100 μg 10μg 50 μg 100 μg Parameter Units IV SC SC SC C₀(IV); C_(max) (SC) μg/mL212 4.95 26.3 50.1 T_(max) hr N/A 24 24 24 t_(1/2, λz) hr 509 ND ND 612AUC_((0-t)) hr · μg/mL 27059 1730 10845 18110 AUC_((0-inf)) hr · μg/mL48269 ND ND 41561 AUC % 43.9 ND ND 56.4 (% extrapolated) V_(ss) (IV); mL1.34 ND ND 2.12 V_(z)/F (SC) Cl (IV); Cl/F mL/hr 0.002 ND ND 0.002 (SC)F (bioavailability) % N/A ND ND 86.1ND: not determinable due to lack of data in the terminal eliminationphase of the concentration versus time profile

Following IV administration, the mAb demonstrated a very low clearance(Cl=0.002 mL/hr) and long elimination half-life (t_(1/2, λz)≈21 days).The mAb demonstrated a steady-state volume of distribution (V_(ss)=1.3mL) that is less than the blood volume in a mouse (≈1.7 mL), suggestingthat the mAb did not distribute substantially out of the vascularcompartment. The back-calculated maximum concentration (C₀) was higherthan expected based on the injected dose and the blood volume in themouse. This, along with the small V_(ss), suggests that the mAb may beconfined, to a large extent, in the serum fraction of the blood.

Following SC administration, C_(max) values increased linearly withdose. At the 100 μg SC dose, the mAb had a t_(1/2, λz) of approximately25 days with clearance and an apparent volume of distribution similar tothat following IV dosing. Bioavailability was 86%. At the lower two SCdoses, most pharmacokinetic parameters could not be estimated due to thelack of a measurable terminal elimination phase, even though sampleswere taken out to 504 hours. The absorption of the mAb following SCdosing appears to reach a steady-state with elimination throughout theduration of the study.

EXAMPLE 31 IL-20 Antagonists in a SCID-hu Transplant Psoriasis Model

Human psoriasis skin grafted on SCID mouse can maintain its clinical,light microscopic, and immunohistochemical psoriatic features forseveral weeks. This model provides a system for evaluating therapiesintended to restore lesional tissue to a normal phenotype. Once thehuman skin is successfully grafted, antibodies against IL-20, IL-20RA,IL-20RB and/or IL-20RA/IL-20RB heterodimers, or soluble IL-20 receptorscan be administered for several weeks, and the epidermal thickness canbe analyzed to evaluate the effect of these antagonists on psoriasis.

Study Design

Full-thickness 6-mm punch biopsies consisting of the entire epidermisand several mm of dermis are obtained healthy adult volunteers andpsoriatic lesional skins. Four to six biopsies are obtained from eachdonor. One punch biopsy from each donor is transplanted onto the dorsalsurface of recipient SCID mouse (CB-17, Taconic). The animals aremaintained in a pathogen-free environment. The treatment is initiatedafter a successful grafting (2-3 weeks post-transplantation) asfollowing: one biopsy for negative control (PBS or isotype mAb), onebiopsy for positive, control (Cyclosporin A), and 2-3 biopsies fortreatment with anti-human IL-20, anti-human IL-20RA, anti-human IL-20RBor anti-human IL-20RA/IL-20RB heterodimer mAb or soluble receptors forIL-20 (intraperitoneal injection, three times a week for 2-4 weeks on aM-W-F schedule).

Quantitative Analysis

Clinical observations and assessments will be made regularly throughoutthe experiments, and will be recorded. The severity of the psoriaticlesions is assessed for scaliness, induration, and erythema in a blindedfashion. The parameters can be scored using the three-point scale:0=complete lack of cutaneous involvement; 1=slight involvement;2=moderate involvement; 3=severe involvement. At the end of the dosingperiod each animal is euthanized and tissues are collected for histologyand IHC. (1) Part of the tissue is fixed in 10% formalin and stainedwith hematoxylin and eosin. Epidermal area is measured as a function ofchanges in epidermal thickness per unit length using NIH Image software.Multiple areas from each transplant are quantified to provide a high nvalue and mean epidermal area. (2) number of inflammatory mononuclearcells per high-power field (0.103×0.135 mm) in the upper dermis; (3) thegrade of parakeratosis is rated on an arbitrary scale from 0 to 3, where0 is no parakeratosis, 1 is parakeratosis in less than one third of thesection, 2 was parakeratosis in more than one third but less than twothirds of the section, a d 3 is parakeratosis in more than two thirds ofthe section. (4) The remaining of the tissue will be stained for Ki67(marker of proliferating keratinocytes), to evaluate the number of Ki67cycling keratinocytes-per-millimeter length of the section. The reducedseverity of psoriasis as measured by epidermal thickness, indicates theneutralization of IL-20 function can be effective in this psoriasismodel. To quantify the reduced severity of psoriasis, we measureepidermal thickness, the number of inflammatory cells in the upperdermis, the numbers of Ki67 cycling keratinocytes, and the grades ofparakeratosis. The significantly reduced all four parameters for thetreated groups compared to the control mice, indicate the potentialtherapeutic use of IL-20 antagonists.

EXAMPLE 32 IL-20 Antagonists in an Organ Culture Psoriasis Model

Human psoriatic plaque skin can be maintained in organ culture, and theabnormal histological features of lesional skin are maintained in theabsence of exogenous growth factors. Antibodies against IL-20, IL-20RA,IL-20RB and/or IL-20RA/IL-20RB heterodimers, or soluble IL-20 receptorscan be administered, and the histological features of psoriatic lesionalskin can be ameliorated.

Study Design

Full-thickness 2-mm punch biopsies consisting of the entire epidermisand several mm of dermis are obtained from either healthy adultvolunteers or from psoriatic lesional skin. Immediately upon biopsy, thetissue is immersed in culture medium consisting of Keratinocyte BasalMedium (KBM) (Clonetics Inc, Walkersville, Md.). The culture medium issupplemented with CaCl2 to bring the final Ca2+ concentration to 1.4 mM(Varani et al, 1993, 1994). The biopsies are then incubated in wells ofa 96-well dish containing 200 ul of Ca2+ supplemented KBM with orwithout additional treatments of antibodies against IL-20, IL-20RA,IL-20RB and/or IL-20RA/IL-20RB heterodimers, or soluble IL-20 receptors.Cultures are incubated at 37° C. in an atmosphere of 95% air and 5% CO₂for 8 days.

Quantitative Analysis

At the end of incubation period, tissue is fixed in 10% bufferedformalin and examined histologically after staining with hematoxylin andeosin. The appearance of psoriatic tissue exposed to the antibodies orsoluble receptors could be more closely resembled that of normaltissues, including the following observation: the initiallydisorganized, irregular-shaped basal epithelial cells developed a morecolumnar appearance with restored polarity; epidermal rete ridgesregressed, with fewer areas of epithelial cell expansion into the dermalspace; and there was less overall degeneration of the upper epidermallayers. The organ culture model provides a rapid and sensitive means fordetermining if a particular compound has potential as ananti-hyperproliferative agent. The abnormal histological feature may beameliorated in the presence of an IL-20antagonist, suggesting theeffectiveness of such agent in the treatment of psoriasis.

EXAMPLE 33 Treatment of Pregnant IL-20 Transgenic Mice with NeutralizingAnti-IL-20, Anti-IL-20RA or Anti-IL-20RB Monoclonal Antibody

To test the rat anti-mouse IL-20, IL-20RA or IL-20RB monoclonalantibodies (mAb) for neutralizing activity in vivo, pregnant IL-20transgenic (Tg) a mice are injected intraperitoneally with one of themAbs listed above. The newborn pups are then assessed for the presenceor absence of the “shiny” skin phenotype that normally characterizesthese strains of mice.

Specifically, male IL-20 Tg (which are generated using the keratin-14 orEulck promoters) mice are bred to C57BL/6 females in estrus and the bredfemales are identified by the presence of a vaginal plug the followingday. Each pregnant female is set aside in a separate cage and monitoreddaily. Treatment groups include at least 4 pregnant females each, toallow for a statistically significant analysis of both Tg and nonTgpups. Based on prior experience with these Tg mice, a litter usuallyranges between approximately 6 to 8 pups per litter, of which between 2to 3 are Tg+.

Seven to nine days after the mice are bred (embryonic age 7-9; e7-9),the females are injected intraperitoneally with 250-500 ug of the mAb(rat IgG2a isotype) in a volume of 200-250 ul of PBS. Short needles areused at a shallow injection angle to avoid directly injecting theuterus. The pregnant females are injected in this manner 3 days a week(Monday, Wednesday, and Friday) for 2 weeks (until birth) in order tosuccessfully access the developing embryos. Control groups (of not lessthan 4 pregnant female mice each) include the following: isotype controlrat IgG2a mAb, anti-human/mouse IL-20 mAb (rat IgG1 isotype), and anisotype control rat IgG1 mAb.

From days 1 through 5 after birth, the pups are closely monitored forthe appearance of the shiny skin phenotype. On day 5, the pups areeuthanized and a portion of the tail is collected for DNA isolation todetermine the genotype (Tg or nonTg) of each pup. Skin samples arecollected for histological analysis in order to assess whether the pupsexhibit the thickened epidermal cell layers that usually characterizethese Tg mice. Trunk blood is also collected from the pups (and aneyebleed from the dams one day after birth) to quantitate, via ELISA,the levels of mAb in the serum of each mouse. Because these mAbs arepotent inhibitors of IL-20 in vivo, the Tg pups have normal skin (i.e.no epidermal thickening or “shiny” appearance).

EXAMPLE 34 Evaluation of Levels of IL-20, IL-20RA and IL-20RB inPsoriatic Skin Samples

Five (5) skin samples with two mice in the control group (CD4+control/CD25+) and three mice in the psoriatic group (CD4+psoriatic/CD25−) were evaluated. Each tissue specimen was fixed inZnTRIS and stained with a mouse anti-human IL-20 monoclonal antibody(clone 240.8.4.7.16.5), mouse anti-human IL-20RA monoclonal antibody(clone HH7.34.1F11.1G2), rat anti-human IL-20RB monoclonal antibody(clone 264.13.1.3.2.3) and rat anti-human IL-22 monoclonal antibody(clone 266.19.1.10.5.2), respectively by immunohistochemistry. Nopositive and negative control cells were applied in this study becausethe fixative used for the cells (10% NBF) was different from the tissues(ZnTRIS). The reagent negative control included mouse isotype IgG andrat isotype IgG to replace the primary antibodies. The stainingintensity of each antibody in the skin samples was outlined in Table 10.TABLE 10 Monoclonal antibodies Isotype IgG Animal/group # TreatmentIL-20 IL-20RA IL-20RB IL-22 Mouse Rat 301 (group 1) CD4+ control/CD25+ −++/b ++/b − − − 307 (group 1) CD4+ control/CD25+ − ++/b ++/b − 315(group 2) CD4+ psoriatic/CD25− − ++++/b ++++/b − 321 (group 2) CD4+psoriatic/CD25− − ++++/b ++++/b − 327 (group 2) CD4+ psoriatic/CD25− −++++/b ++++/b − − −±, +, ++, +++ & ++++: Staining intensity from weak to strong; −: Nostaining; b: Background staining.Human IL-20 Monoclonal Antibody (Clone 240.8.4.7.16.5)

IL-20 was not detected in either the control or psoriatic skin samples.

Mouse Anti-Human IL-20RA Monoclonal Antibody (Clone HH7.34.1F11.1G2)

High expression of IL-20RA was observed in the psoriatic skin samples(group 2) compared to the controls (group 1). In the control group,epidermis and few scattered mononuclear cells in the dermis displayedpositive staining. In the psoriatic group, the tissues were stained inthe same manner but at a higher level, e.g., strong positive stainingwas observed in the epidermis and on large number infiltratedmononuclear cells in the dermis. The skin demonstrated psoriasis-likedermatitis and the IL-20RA staining in the epidermis was mainly locatedin the outer layers (stratum granulosum and stratum corneum). Stratumbasale, the germinal layer of the epidermis showed no staining andstratum spinosum, the prickle layer of the epidermis showed weak anddiffused staining. The antibody also showed some non-specific stainingin the connective tissues. Skin samples stained with mouse isotype IgGshowed negative staining.

Rat Anti-Human IL-20RB Monoclonal Antibody (Clone 264.13.1.3.2.3)

The rat anti-human IL-20RB monoclonal antibody demonstrated a similarstaining as the mouse anti-human IL-20RA monoclonal antibody but withfew positive mononuclear cells in the dermis. The antibody also showednon-specific staining to skeletal muscle. Skin samples stained with ratisotype IgG exhibited negative staining

Rat Anti-Human IL-22 Monoclonal Antibody (Clone 266.19.1.10.5.2)

IL-22 was not detected in either the control or psoriatic skin samples.

Conclusion

IL-20RA and IL-20RB expression were observed in the epidermis in thepsoriatic skin samples (CD4+ psoratic/CD25−) by immunohistochemistry andmicroscopically these tissues exhibited abnormally thickened epidermisand severe dermatitis. The vast majority of the IL-20RA and IL-20RBexpression was found in the epidermis appeared to be in thekeratinocytes above the basal and prickle cell layers mainly in thestratum granulosum characterized by intracellular granules whichcontributed to the process of keratinisation and in the stratum corneumconsisted of flattened, fused cell remnants (keratine). In addition,IL-20RA expression was also observed on mononuclear cells in the areawith dermatitis. The control tissues (CD4+/control/CD25+) demonstratedrelatively low level of IL-20RA and IL-20RB expression compared with thepsoriatic tissues. No IL-20 and IL-22 ligand were detected in either thecontrol or psoriatic skin samples using the human IL-20 monoclonalantibody and the rat anti-human IL-22 monoclonal antibody. All the skinsamples stained with mouse isotype IgG or rat isotype IgG showednegative staining.

EXAMPLE 35 IL-20RA Expression in Skin Samples from IL-20TG/IL-20RAKnock-Out Mice

Eight (8) neonates with two (2) neonates in each genotype group: TG/0HOM, TG/0 Het, 0/0 HOM and 0/0 Het from the K14 IL-20 m (TG)/IL-20RA(KO) were studied. The caudal thorax from each animal was fixed in 10%neutral buffered formalin (NBF) and stained with a mouse anti-humanIL-20RA monoclonal antibody (clone HH7.34.1F11.1G2) byimmunohistochemistry (IL-20RA-IHC#15, ARK IHC protocol). Baf3 cellstransfected with either human or murine IL-20RA/RB and human lung knownwith IL-20RA expression were used as positive controls. The reagentnegative control included mouse isotype IgG to replace the primaryantibody.

IHC Results

A. The Positive Control Cells and Tissues

Scattered mononuclear cells in the human lung demonstrated weakstaining, however, the Baf3 cells transfected with murine IL-20RA/RBdisplayed negative staining. The Baf3 cells transfected with humanIL-20RA/RB were not found on the slide, which might due to a poor samplepreparation or cell loss by repeat buffer washing during IHC procedure.The wild type Baf3 cells were clear with no staining.

B. The K14 IL20m (TG)/IL-20RA (KO) Neonate Skins

Weak expression of IL-20RA was found in one out of two TG/0 Het and oneout of two 0/0 Het neonate skins, e.g., epidermal cells above the basallayer in the skin showed diffused cytoplasmic staining with themonoclonal antibody. The skins from TG/0 HOM (n=2) and 0/0 HOM (n=2)neonates showed no IL-20RA expression. All the neonate skins (TG/0 HOM,TG/0 Het, 0/0 HOM and 0/0 Het) stained with mouse isotype IgG showednegative staining. The staining intensity of IL-20RA was outlined inTable 11. TABLE 11 Genotype Animal number Epidermal thickness IL-20RAexpression TG/0 HOM 36109 44.28 μm − TG/0 HOM 36242 39.01 μm − TG/0 Het36020 40.90 μm − TG/0 Het 36241 39.63 μm ± 0/0 HOM 36130 30.05 μm − 0/0HOM 36176 29.44 μm − 0/0 Het 36128 30.72 μm − 0/0 Het 36245 30.05 μm ±±, +, ++, +++ & ++++: Staining intensity from weak to strong;−: No staining.Conclusion

IL-20RA expression was observed in the epidermal cells in the TG/0 Het(IL-20TG×IL-20RA KO) neonates by immunohistochemistry as describedabove. Physioscreen studies revealed that these neonates grosslyexhibited shiny skin phenotype and microscopically exhibited abnormallythickened epidermis. Further, IL-20RA mAb staining was associated withepidermal thickening. The IHC results revealed that one out of twoneonate skins in TG/0 Het and 0/0 Het showed positive staining ofIL-20RA, respectively. The vast majority of the IL-20RA expressing cellsin the epidermis appeared to be in the keratinocytes above the basallayer (suprabasal), but the staining was relatively weaker compared tothe skin samples of TG/0 Het from the IL-20 TG×IL-20RA KO study. NoIL-20RA expression was observed in the epidermis in either the TG/0 HOMor 0/0 HOM. In sum, IL-20RA mAb staining was positive in Het but not inHOM.

EXAMPLE 36 IL-20RA Expression in Skin Samples from IL-20TG/IL-20RAKnock-Out Mice

Eight (8) neonates with two (2) neonates in each genotype group: TG/−HOM, TG/− Het, −/− HOM and −/− Het from the K14 IL-20m (TG)/IL-22RA (KO)were studied. The caudal thorax from each animal was fixed in 10%neutral buffered formalin (NBF) and stained with a mouse anti-humanIL-20RA monoclonal antibody raised against human (cloneHH7.34.1F11.1G2). Cos cells transfected with human IL-20RA and humanlung known with IL-20RA expression was used as positive controls.However, cells and tissues expressing mouse IL-20RA were not available,as the control because the IL-20RA TG mice died before birth. Thereagent negative control included mouse isotype IgG to replace theprimary antibody.

IHC Results

A. The Positive Control Tissue (Human Lung)

Scattered mononuclear cells in the human lung demonstrated positivestaining.

B. The Mouse Tissues (IL-20TG/IL-22RA KO Neonate Skins)

The anti-IL-20RA MAb recognized mouse IL-20RA in IHC. Expression ofIL-20RA was found in IL-20TG/IL-20RA KO Het neonate skins (n=2) usingIHC, e.g., epidermal cells in the skin showed diffused cytoplasmicstaining with the antibody. The skins from TG/− HOM (n=2), −/− HOM (n=2)and −/− Het (n=2) neonates showed no IL-20RA expression. The TG/Hetneonate skin stained with mouse isotype IgG showed negative staining.

Conclusion

IL-20RA expression was observed in the epidermal cells in the TG/− Hetneonates by immunohistochemistry and previous physioscreen studiesrevealed that these neonates grossly exhibited shiny skin phenotype andmicroscopically exhibited abnormally thickened epidermis. The vastmajority of the IL-20RA expressing cells in the epidermis appeared to bein the keratinocytes above the basal layer (suprabasal) and the stainingwas not observed in the other tissues in the section. No L-20RAexpression was observed in the epidermis in either of the TG/− HOMneonates or the non-UL-20 TG neonates (−/− HOM and −/− Het) thatdisplayed no shiny phenotype. Further, all the skin samples stained withmouse isotype IgG showed negative staining.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method of producing an antibody to a polypeptide comprising:inoculating an animal with a polypeptide selected from the groupconsisting of: a) a polypeptide consisting of amino acid residues 42(Ile) to 102 (Asp) of SEQ ID NO:2; b) a polypeptide consisting of aminoacid residues 42 (Ile) to 60 (Ile) of SEQ ID NO:2; c) a polypeptideconsisting of amino acid residues 42 (Ile) to 69 (Glu) of SEQ ID NO:2;d) a polypeptide consisting of amino acid residues 42 (Ile) to 81 (Cys)of SEQ ID NO:2; e) a polypeptide consisting of amino acid residues 42(Ile) to 96 (Lys) of SEQ ID NO:2; f) a polypeptide consisting of aminoacid residues 42 (Ile) to 102 (Asp) of SEQ ID NO:2; g) a polypeptideconsisting of amino acid residues 60 (Ile) to 69 (Glu) of SEQ ID NO:2;h) a polypeptide consisting of amino acid residues 60 (Ile) to 81 (Cys)of SEQ ID NO:2; i) a polypeptide consisting of amino acid residues 60(Ile) to 96 (Lys) of SEQ ID NO:2; j) a polypeptide consisting of aminoacid residues 60 (Ile) to 102 (Asp) of SEQ ID NO:2; k) a polypeptideconsisting of amino acid residues 69 (Glu) to 81 (Cys) of SEQ ID NO:2;l) a polypeptide consisting of amino acid residues 69 (Glu) to 96 (Lys)of SEQ ID NO:2; m) a polypeptide consisting of amino acid residues 69(Glu) to 102 (Asp) of SEQ ID NO:2; n) a polypeptide consisting of aminoacid residues 81 (Cys) to 96 (Lys) of SEQ ID NO:2; o) a polypeptideconsisting of amino acid residues 81 (Cys) to 102 (Asp) of SEQ ID NO:2;and p) a polypeptide consisting of amino acid residues 96 (Lys) to 102(Asp) of SEQ ID NO:2, wherein the polypeptide elicits an immune responsein the animal to produce the antibody; and isolating the antibody fromthe animal; and wherein the antibody specifically binds to an IL-20polypeptide (SEQ ID NO:2 or SEQ ID NO:3).
 2. The method of claim 1,wherein the antibody produced by the method inhibits thepro-inflammatory activity of IL-20.
 3. The method of claim 1, whereinthe antibody produced by the method neutralizes the interaction of IL-20with its receptor.
 4. The method of claim 3, wherein the neutralizationby the antibody is measured by showing neutralization in an in vitrocell-based assay.
 5. The method of claim 1, wherein the antibodyproduced by the method reduces the pro-inflammatory activity of IL-20.6. An antibody produced by the method of claim 1, which binds to apolypeptide of SEQ ID NO:2 or SEQ ID NO:3.
 7. The antibody of claim 6,wherein the antibody is selected from the group consisting of: (a) apolyclonal antibody, (b) a murine monoclonal antibody, (c) a humanizedantibody derived from (b), (d) an antibody fragment, and (e) a humanmonoclonal antibody.
 8. The antibody of claim 7, wherein the antibodyfurther comprises a radionuclide, enzyme, substrate, cofactor,fluorescent marker, chemiluminescent marker, peptide tag, magneticparticle, drug, or toxin.
 9. The antibody of claim 7, wherein theantibody further comprises PEGylation.
 10. An antibody or antibodyfragment that binds to a polypeptide comprising a sequence of amino acidresidues as shown in SEQ ID NO:2; wherein the antibody or antibodyfragment inhibits the pro-inflammatory activity of IL-20.
 11. Anantibody or antibody fragment that binds to a polypeptide comprising asequence of amino acid residues as shown in SEQ ID NO:2; wherein theantibody or antibody fragment reduces the pro-inflammatory activity ofIL-20.
 12. The antibody of claim 10, wherein the antibody furthercomprises a radionuclide, enzyme, substrate, cofactor, fluorescentmarker, chemiluminescent marker, peptide tag, magnetic particle, drug,or toxin.
 13. The antibody of claim 10, wherein the antibody furthercomprises PEGylation.
 14. The antibody of claim 11, wherein the antibodyfurther comprises a radionuclide, enzyme, substrate, cofactor,fluorescent marker, chemiluminescent marker, peptide tag, magneticparticle, drug, or toxin.
 15. The antibody of claim 11, wherein theantibody further comprises PEGylation.
 16. A method for reducing orinhibiting IL-20-induced proliferation or differentiation ofhematopoietic cells and hematopoietic cell progenitors comprisingculturing bone marrow or peripheral blood cells with a compositioncomprising an amount of an antibody according to claim 3 sufficient toreduce proliferation or differentiation of the hematopoietic cells inthe bone marrow or peripheral blood cells as compared to bone marrow orperipheral blood cells cultured in the absence of soluble cytokinereceptor.
 17. The method of claim 16, wherein the hematopoietic cellsand hematopoietic progenitor cells are lymphoid cells.
 18. The method ofclaim 17, wherein the lymphoid cells are macrophages or T cells.
 19. Amethod of reducing IL-20-induced inflammation comprising administeringto a mammal with inflammation an amount of a composition comprising anantibody according to claim 3, wherein the amount of the compositionadministered is sufficient to reduce inflammation.
 20. A method ofreducing IL-20-induced inflammation comprising administering to a mammalwith inflammation an amount of a composition comprising an antibody orantibody fragment according to claim 10, wherein the amount of thecomposition administered is sufficient to reduce inflammation.
 21. Amethod of suppressing an inflammatory response in a mammal withinflammation comprising: (1) determining a level of serum amyloid Aprotein; (2) administering a composition comprising an antibodyaccording to claim 3 in an acceptable pharmaceutical vehicle; (3)determining a post administration level of serum amyloid A protein; (4)comparing the level of serum amyloid A protein in step (1) to the levelof serum amyloid A protein in step (3), wherein a lack of increase or adecrease in serum amyloid A protein level is indicative of suppressingan inflammatory response.
 22. An antibody comprising a monoclonalantibody that that binds to an epitope of human IL-20 (SEQ ID NO:2 or3), wherein the epitope is selected from the group consisting of: (a) apolypeptide consisting of the amino acid sequence of SEQ ID NO:2 fromamino acid residues 42 (Ile) to 102 (Asp); (b) a polypeptide consistingof the amino acid sequence of SEQ ID NO:2 from amino acid residues 42(Ile) to 60 (Ile); (c) a polypeptide consisting of the amino acidsequence of SEQ ID NO:2 from amino acid residues 42 (Ile) to 69 (Glu);(d) a polypeptide consisting of the amino acid sequence of SEQ ID NO:2from amino acid residues 42 (Ile) to 81 (Cys); (e) a polypeptideconsisting of the amino acid sequence of SEQ ID NO:2 from amino acidresidues 42 (Ile) to 96 (Lys); (f) a polypeptide consisting of the aminoacid sequence of SEQ ID NO:2 from amino acid residues 42 (Ile) to 102(Asp); (g) a polypeptide consisting of the amino acid sequence of SEQ IDNO:2 from amino acid residues 60 (Ile) to 69 (Glu); (h) a polypeptideconsisting of the amino acid sequence of SEQ ID NO:2 from amino acidresidues 60 (Ile) to 81 (Cys); (i) a polypeptide consisting of the aminoacid sequence of SEQ ID NO:2 from amino acid residues 60 (Ile) to 96(Lys); (j) a polypeptide consisting of the amino acid sequence of SEQ IDNO:2 from amino acid residues 60 (Ile) to 102 (Asp); (k) a polypeptideconsisting of the amino acid sequence of SEQ ID NO:2 from amino acidresidues 69 (Glu) to 81 (Cys); (l) a polypeptide consisting of the aminoacid sequence of SEQ ID NO:2 from amino acid residues 69 (Glu) to 96(Lys); (m) a polypeptide consisting of the amino acid sequence of SEQ IDNO:2 from amino acid residues 69 (Glu) to 102 (Asp); (n) a polypeptideconsisting of the amino acid sequence of SEQ ID NO:2 from amino acidresidues 81 (Cys) to 96 (Lys); (o) a polypeptide consisting of the aminoacid sequence of SEQ ID NO:2 from amino acid residues 81 (Cys) to 102(Asp); and (p) a polypeptide consisting of the amino acid sequence ofSEQ ID NO:2 from amino acid residues 96 (Lys) to 102 (Asp) of SEQ IDNO:2. wherein the antibody reduces or neutralizes the pro-inflammatoryactivity of human IL-20 (SEQ ID NO:8)
 23. The antibody of claim 22,wherein the antibody further comprises a radionuclide, enzyme,substrate, cofactor, fluorescent marker, chemiluminescent marker,peptide tag, magnetic particle, drug, or toxin.
 24. The antibody ofclaim 22, wherein the antibody is selected from the group consisting of:(a) a murine monoclonal antibody, (b) a humanized antibody derived from(a), (c) an antibody fragment, and (d) a human monoclonal antibody. 25.A method of treating a pathological condition in a subject associatedwith IL-20 activity comprising administering an effective amount of theantibody of claim 22, thereby treating said pathological condition. 26.The method of claim 25, wherein said pathological condition is a chronicinflammatory condition.
 27. The method of claim 26, wherein said chronicinflammatory condition is selected from the group consisting of: (a)inflammatory bowel disease; (b) ulcerative colitis; (c) Crohn's disease;(d) arthritis; and (e) psoriasis.
 28. The method of claim 25, whereinsaid pathological condition is an acute inflammatory condition.
 29. Themethod of claim 28, wherein said acute inflammatory condition isselected from the group consisting of: (a) endotoxemia; (b) septicemia;(c) toxic shock syndrome; and (d) infectious disease.
 30. A method oftreating a mammal afflicted with an inflammatory disease in which IL-20plays a role, comprising: administering an antagonist of IL-20 to themammal such that the inflammation is reduced, wherein the antagonistcomprises (i) an antibody, antibody fragment, or binding polypeptidethat specifically binds a polypeptide or polypeptide fragment of IL-20or (ii) a polypeptide or polypeptide fragment of IL-20; and wherein theinflammatory activity of IL-20 is reduced.
 31. The method of claim 30,wherein the disease is a chronic inflammatory disease.
 32. The method ofclaim 31, wherein the disease is a chronic inflammatory diseasecomprising inflammatory bowel disease, ulcerative colitis, Crohn'sdisease, arthritis, atopic dermatitis, or psoriasis.
 33. The method ofclaim 30, wherein the disease is an acute inflammatory disease.
 34. Themethod of claim 33, wherein the disease is an acute inflammatory diseasecomprising endotoxemia, septicemia, toxic shock syndrome or infectiousdisease.
 35. The method of claim 30, wherein the antibody is selectedfrom the group consisting of: (a) a polyclonal antibody, (b) a murinemonoclonal antibody, (c) a humanized antibody derived from (b), (d) anantibody fragment, and (e) a human monoclonal antibody.
 36. The methodof claim 30, wherein the antibody, antibody fragment, or bindingpolypeptide further comprises a radionuclide, enzyme, substrate,cofactor, fluorescent marker, chemiluminescent marker, peptide tag,magnetic particle, drug, or toxin.
 37. The method of claim 30, whereinthe antibody further comprises PEGylation.
 38. A method of reducinginflammation comprising administering to a mammal with inflammation anamount of a composition comprising an antibody according to claim 22,wherein the amount of the composition administered is sufficient toreduce inflammation.